case study animal testing

Animal Research

Animal research case studies

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

Dr Clare Stanford: using mice to find treatments for ADHD

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

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

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

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

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

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

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

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

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

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

Watch CBS News

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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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.

Organizations and Resources

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

• Learn more about outreach and advocacy organizations

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. 

• Learn more about research discoveries at Stanford

Undercover investigation exposes the horrors of animal testing—and more than 80 dogs who need our help

Kitty Block and Sara Amundson

Today we are releasing the results of our seven-month undercover investigation at one of America’s largest animal testing laboratories. We’re asking you to join us in changing an outdated industry— animal testing—and, more immediately, in urging the release of more than 80 dogs still suffering at the lab.

Our undercover investigator worked on studies with more than 6,000 animals—including 250 dogs, 500 primates, 62 “minipigs” and more than 5,100 mice and rats—at Inotiv, a contract testing laboratory in Indiana. Inotiv has laboratories and breeding facilities in multiple states, including Colorado, Maryland, North Carolina, Texas and Virginia. What we saw was heartbreaking. Our investigator documented animals being force-fed high doses of drugs via tubes or intravenously, sometimes several times a day. Some animals were unable to move because of the drugs’ toxic effects; others died during procedures. The studies conducted at Inotiv were intended to test drug toxicity and were funded by dozens of pharmaceutical companies.

Most of the animals our investigator came to know didn’t make it out alive, including one beagle we call Riley, because we believe he deserves a name and not just a number. He was used to test a substance so toxic that it brought him near death after only two days of dosing. The investigator captured video showing Riley hypersalivating, trembling, vomiting and moaning on the floor, unable to stand. Our investigator tried to comfort him while he was dying, but Riley was left to suffer overnight because the veterinarian was unavailable on a weekend evening. Riley was euthanized the following day.

Other animals who died horrible deaths during our investigation were two young cynomolgus macaques who were accidentally hanged in their restraint chairs.

We documented numerous potential violations of the Animal Welfare Act, including failure to provide adequate veterinary care, failure to minimize animal pain and distress, and failure to ensure proper staffing. We urgently reached out to the U.S. Department of Agriculture and asked the agency to investigate these findings, but the devastating truth is that this suffering—and Riley’s fate—are simply reflections of the realities of contemporary animal testing.

Pharmaceutical companies and the Food and Drug Administration have a responsibility to ensure drugs are safe for humans, but our continuing reliance on animal testing creates a false sense of security. Nearly 90% of drugs tested in animals ultimately fail in human trials , and a large proportion of these failures is attributable to unexpected toxicity in humans … even after animal tests. In what business model is such a high failure rate acceptable?

We uncovered an example of this failure. A company contracting with Inotiv ended its pursuit of a drug after encountering unexpected liver toxicity in human patients. At the same time the company decided to end the human trials, mice and primates were being dosed with the same drug at Inotiv while our investigator was there. We are hoping these tests have by now come to an end since the pharmaceutical company has stopped pursuit of this compound due to the serious adverse effects in humans.

Today, combinations of modern, non-animal methods based on human biology—such as human organs on chips and next-generation computer modeling—are increasingly providing better real-world predictions of human reactions to drugs and chemicals than some animal tests. For example, a recent study found that organ chips detected toxicity in almost seven out of eight drugs that proved toxic in patients but had cleared animal testing—an 87% success rate. But because animal testing remains the surest path to regulatory approval, it continues.

We ask that you join us in bringing the science of safety testing into the 21 st century—bringing the system from low-tech and cruel to high-tech and effective. Even more urgently, we’re asking for your help in securing the release of 82 dogs still alive in the lab today.

Eighty beagle puppies are being dosed every day and may suffer the same fate as Riley if we don’t act quickly. Two additional adult beagles have been used to practice harmful procedures for years. Please join us in urging Inotiv (the testing laboratory) and Crinetics (the pharmaceutical company that hired Inotiv) to immediately end the tests and release these 82 beagles to us so that we can find them loving homes.

With your help, we’ll continue our work toward the day when invasive experiments on dogs and other animals are a thing of the past—and saving these dogs is just the start.

Help us secure the release of the 82 dogs still alive in the lab

Sara Amundson is president of the Humane Society Legislative Fund.

Taking Suffering Out of Science

About the author

Kitty Block is President and CEO of the Humane Society of the United States and CEO of Humane Society International, the international affiliate of the HSUS

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Also of interest

Our undercover investigation reveals why hippos desperately need federal protections under the Endangered Species Act

South Korea shuts down major dog meat market; More than 80 dogs rescued from vendors

Undercover investigation exposes Petland's treatment of sick puppies

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Open Access

Ethical and Scientific Considerations Regarding Animal Testing and Research

* E-mail: [email protected]

Affiliations Physicians Committee for Responsible Medicine, Washington, D.C., United States of America, Department of Medicine, The George Washington University, Washington, D.C., United States of America

Affiliation Physicians Committee for Responsible Medicine, Washington, D.C., United States of America

• Hope R. Ferdowsian, 

Published: September 7, 2011

• https://doi.org/10.1371/journal.pone.0024059
• Reader Comments

Citation: Ferdowsian HR, Beck N (2011) Ethical and Scientific Considerations Regarding Animal Testing and Research. PLoS ONE 6(9): e24059. https://doi.org/10.1371/journal.pone.0024059

Editor: Catriona J. MacCallum, Public Library of Science, United Kingdom

Copyright: © 2011 Ferdowsian, Beck. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The authors are grateful to the National Science Foundation (grant SES-0957163) and the Arcus Foundation (grant 0902-34) for the financial support for the corresponding conference, Animals, Research, and Alternatives: Measuring Progress 50 Years Later. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: HRF and NB are employed by Physicians Committee for Responsible Medicine, which is a non-governmental organization which promotes higher ethical standards in research and alternatives to the use of animals in research, education, and training. Physicians Committee for Responsible Medicine is a nonprofit organization, and the authors adhered to PLoS ONE policies on sharing data and materials.

In 1959, William Russell and Rex Burch published the seminal book, The Principles of Humane Experimental Technique, which emphasized r eduction, r efinement, and r eplacement of animal use, principles which have since been referred to as the “3 Rs”. These principles encouraged researchers to work to reduce the number of animals used in experiments to the minimum considered necessary, refine or limit the pain and distress to which animals are exposed, and replace the use of animals with non-animal alternatives when possible. Despite the attention brought to this issue by Russell and Burch and since, the number of animals used in research and testing has continued to increase, raising serious ethical and scientific issues. Further, while the “3 Rs” capture crucially important concepts, they do not adequately reflect the substantial developments in our new knowledge about the cognitive and emotional capabilities of animals, the individual interests of animals, or an updated understanding of potential harms associated with animal research. This Overview provides a brief summary of the ethical and scientific considerations regarding the use of animals in research and testing, and accompanies a Collection entitled Animals, Research, and Alternatives: Measuring Progress 50 Years Later , which aims to spur ethical and scientific advancement.

Introduction

One of the most influential attempts to examine and affect the use of animals in research can be traced back to1959, with the publication of The Principles of Humane Experimental Technique [1] . William Russell and Rex Burch published this seminal book in response to marked growth in medical and veterinary research and the concomitant increase in the numbers of animals used. Russell and Burch's text emphasized r eduction, r efinement, and r eplacement of animal use, principles which have since been referred to as the “3 Rs”. These principles encouraged researchers to work to reduce the number of animals used in experiments to the minimum considered necessary, refine or limit the pain and distress to which animals are exposed, and replace the use of animals with non-animal alternatives when possible.

Despite the attention brought to this issue by Russell and Burch, the number of animals used in research and testing has continued to increase. Recent estimates suggest that at least 100 million animals are used each year worldwide [2] . However, this is likely an underestimate, and it is impossible to accurately quantify the number of animals used in or for experimentation. Full reporting of all animal use is not required or made public in most countries. Nevertheless, based on available information, it is clear that the number of animals used in research has not significantly declined over the past several decades.

The “3 Rs” serve as the cornerstone for current animal research guidelines, but questions remain about the adequacy of existing guidelines and whether researchers, review boards, and funders have fully and adequately implemented the “3 Rs”. Further, while the “3 Rs” capture crucially important concepts, they do not adequately reflect the substantial developments in our new knowledge about the cognitive and emotional capabilities of animals; an updated understanding of the harms inherent in animal research; and the changing cultural perspectives about the place of animals in society [3] , [4] . In addition, serious questions have been raised about the effectiveness of animal testing and research in predicting anticipated outcomes [5] – [13] .

In August 2010, the Georgetown University Kennedy Institute of Ethics, the Johns Hopkins University Center for Alternatives to Animal Testing, the Institute for In Vitro Sciences, The George Washington University, and the Physicians Committee for Responsible Medicine jointly held a two day multi-disciplinary, international conference in Washington, DC, to address the scientific, legal, and political opportunities and challenges to implementing alternatives to animal research. This two-day symposium aimed to advance the study of the ethical and scientific issues surrounding the use of animals in testing and research, with particular emphasis on the adequacy of current protections and the promise and challenges of developing alternatives to the use of animals in basic research, pharmaceutical research and development, and regulatory toxicology. Speakers who contributed to the conference reviewed and contributed new knowledge regarding the cognitive and affective capabilities of animals, revealed through ethology, cognitive psychology, neuroscience, and related disciplines. Speakers also explored the dimensions of harm associated with animal research, touching on the ethical implications regarding the use of animals in research. Finally, several contributors presented the latest scientific advances in developing alternatives to the use of animals in pharmaceutical research and development and regulatory toxicity testing.

This Collection combines some papers that were written following this conference with an aim to highlight relevant progress and research. This Overview provides a brief summary of the ethical and scientific considerations regarding the use of animals in research and testing, some of which are highlighted in the accompanying Collection.

Analysis and Discussion

Ethical considerations and advances in the understanding of animal cognition.

Apprehension around burgeoning medical research in the late 1800s and the first half of the 20 th century sparked concerns over the use of humans and animals in research [14] , [15] . Suspicions around the use of humans were deepened with the revelation of several exploitive research projects, including a series of medical experiments on large numbers of prisoners by the Nazi German regime during World War II and the Tuskegee syphilis study. These abuses served as the impetus for the establishment of the Nuremberg Code, Declaration of Helsinki, and the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research (1974) and the resulting Belmont Report [16] – [18] . Today, these guidelines provide a platform for the protection of human research subjects, including the principles of respect, beneficence, and justice, as well as special protections for vulnerable populations.

Laws to protect animals in research have also been established. The British Parliament passed the first set of protections for animals in 1876, with the Cruelty to Animals Act [19] . Approximately ninety years later, the U.S. adopted regulations for animals used in research, with the passage of the Laboratory Animal Welfare Act of 1966 [20] . Subsequent national and international laws and guidelines have provided basic protections, but there are some significant inconsistencies among current regulations [21] . For example, the U.S. Animal Welfare Act excludes purpose-bred birds, rats, or mice, which comprise more than 90% of animals used in research [20] . In contrast, certain dogs and cats have received special attention and protections. Whereas the U.S. Animal Welfare Act excludes birds, rats and mice, the U.S. guidelines overseeing research conducted with federal funding includes protections for all vertebrates [22] , [23] . The lack of consistency is further illustrated by the “U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training” which stress compliance with the U.S. Animal Welfare Act and “other applicable Federal laws, guidelines, and policies” [24] .

While strides have been made in the protection of both human and animal research subjects, the nature of these protections is markedly different. Human research protections emphasize specific principles aimed at protecting the interests of individuals and populations, sometimes to the detriment of the scientific question. This differs significantly from animal research guidelines, where the importance of the scientific question being researched commonly takes precedence over the interests of individual animals. Although scientists and ethicists have published numerous articles relevant to the ethics of animal research, current animal research guidelines do not articulate the rationale for the central differences between human and animal research guidelines. Currently, the majority of guidelines operate on the presumption that animal research should proceed based on broad, perceived benefits to humans. These guidelines are generally permissive of animal research independent of the costs to the individual animal as long as benefits seem achievable.

The concept of costs to individual animals can be further examined through the growing body of research on animal emotion and cognition. Studies published in the last few decades have dramatically increased our understanding of animal sentience, suggesting that animals' potential for experiencing harm is greater than has been appreciated and that current protections need to be reconsidered. It is now widely acknowledged by scientists and ethicists that animals can experience pain and distress [25] – [29] . Potential causes of harm include invasive procedures, disease, and deprivation of basic physiological needs. Other sources of harm for many animals include social deprivation and loss of the ability to fulfill natural behaviors, among other factors. Numerous studies have demonstrated that, even in response to gentle handling, animals can show marked changes in physiological and hormonal markers of stress [30] .

Although pain and suffering are subjective experiences, studies from multiple disciplines provide objective evidence of animals' abilities to experience pain. Animals demonstrate coordinated responses to pain and many emotional states that are similar to those exhibited by humans [25] , [26] . Animals share genetic, neuroanatomical, and physiological similarities with humans, and many animals express pain in ways similar to humans. Animals also share similarities with humans in genetic, developmental, and environmental risk factors for psychopathology [25] , [26] . For example, fear operates in a less organized subcortical neural circuit than pain, and it has been described in a wide variety of species [31] . More complex markers of psychological distress have also been described in animals. Varying forms of depression have been repeatedly reported in animals, including nonhuman primates, dogs, pigs, cats, birds and rodents, among others [32] – [34] . Anxiety disorders, such as post-traumatic stress disorder, have been described in animals including chimpanzees and elephants [35] , [36] , [37] .

In addition to the capacity to experience physical and psychological pain or distress, animals also display many language-like abilities, complex problem-solving skills, tool related cognition and pleasure-seeking, with empathy and self-awareness also suggested by some research. [38] – [44] . Play behavior, an indicator of pleasure, is widespread in mammals, and has also been described in birds [45] , [46] . Behavior suggestive of play has been observed in other taxa, including reptiles, fishes and cephalopods [43] . Self-awareness, assessed through mirror self-recognition, has been reported for chimpanzees and other great apes, magpies, and some cetaceans. More recent studies have shown that crows are capable of creating and using tools that require access to episodic-like memory formation and retrieval [47] . These findings suggest that crows and related species display evidence of causal reasoning, flexible learning strategies, imagination and prospection, similar to findings in great apes. These findings also challenge our assumptions about species similarities and differences and their relevance in solving ethical dilemmas regarding the use of animals in research.

Predictive Value of Animal Data and the Impact of Technical Innovations on Animal Use

In the last decade, concerns have mounted about how relevant animal experiments are to human health outcomes. Several papers have examined the concordance between animal and human data, demonstrating that findings in animals were not reliably replicated in human clinical research [5] – [13] . Recent systematic reviews of treatments for various clinical conditions demonstrated that animal studies have been poorly predictive of human outcomes in the fields of neurology and vascular disease, among others [7] , [48] . These reviews have raised questions about whether human diseases inflicted upon animals sufficiently mimic the disease processes and treatment responses seen in humans.

The value of animal use for predicting human outcomes has also been questioned in the regulatory toxicology field, which relies on a codified set of highly standardized animal experiments for assessing various types of toxicity. Despite serious shortcomings for many of these assays, most of which are 50 to 60 years old, the field has been slow to adopt newer methods. The year 2007 marked a turning point in the toxicology field, with publication of a landmark report by the U.S. National Research Council (NRC), highlighting the need to embrace in vitro and computational methods in order to obtain data that more accurately predicts toxic effects in humans. The report, “Toxicity Testing in the 21 st Century: A Vision and a Strategy,” was commissioned by the U.S. Environmental Protection Agency, partially due to the recognition of weaknesses in existing approaches to toxicity testing [49] . The NRC vision calls for a shift away from animal use in chemical testing toward computational models and high-throughput and high-content in vitro methods. The report emphasized that these methods can provide more predictive data, more quickly and affordably than traditional in vivo methods. Subsequently published articles address the implementation of this vision for improving the current system of chemical testing and assessment [50] , [51] .

While a sea change is underway in regulatory toxicology, there has been much less dialogue surrounding the replacement of animals in research, despite the fact that far more animals are used in basic and applied research than in regulatory toxicology. The use of animals in research is inherently more difficult to approach systematically because research questions are much more diverse and less proscribed than in regulatory toxicology [52] . Because researchers often use very specialized assays and systems to address their hypotheses, replacement of animals in this area is a more individualized endeavour. Researchers and oversight boards have to evaluate the relevance of the research question and whether the tools of modern molecular and cell biology, genetics, biochemistry, and computational biology can be used in lieu of animals. While none of these tools on their own are capable of replicating a whole organism, they do provide a mechanistic understanding of molecular events. It is important for researchers and reviewers to assess differences in the clinical presentation and manifestation of diseases among species, as well as anatomical, physiological, and genetic differences that could impact the transferability of findings. Another relevant consideration is how well animal data can mirror relevant epigenetic effects and human genetic variability.

Examples of existing and promising non-animal methods have been reviewed recently by Langley and colleagues, who highlighted advances in fields including orthodontics, neurology, immunology, infectious diseases, pulmonology, endocrine and metabolism, cardiology, and obstetrics [52] .

Many researchers have also begun to rely solely on human data and cell and tissue assays to address large areas of therapeutic research and development. In the area of vaccine testing and development, a surrogate in-vitro human immune system has been developed to help predict an individual's immune response to a particular drug or vaccine [53] , [54] . This system includes a blood-donor base of hundreds of individuals from diverse populations and offers many benefits, including predictive high-throughput in vitro immunology to assess novel drug and vaccine candidates, measurement of immune responses in diverse human populations, faster cycle time for discovery, better selection of drug candidates for clinical evaluation, and reductions in the time and costs to bring drugs and vaccines to the market. In the case of vaccines, this system can be used at every stage, including in vitro disease models, antigen selection and adjuvant effects, safety testing, clinical trials, manufacturing, and potency assays. When compared with data from animal experiments, this system has produced more accurate pre-clinical data.

The examples above illustrate how innovative applications of technology can generate data more meaningful to humans, and reduce or replace animal use, but advances in medicine may also require novel approaches to setting research priorities. The Dr. Susan Love Research Foundation, which focuses on eradicating breast cancer, has challenged research scientists to move from animal research to breast cancer prevention research involving women. If researchers could better understand the factors that increase the risk for breast cancer, as well as methods for effective prevention, fewer women would require treatment for breast cancer. Whereas animal research is largely investigator-initiated, this model tries to address the questions that are central to the care of women at risk for or affected by breast cancer. This approach has facilitated the recruitment of women for studies including a national project funded by the National Institutes of Health and the National Institute of Environmental Health to examine how environment and genes affect breast cancer risk. This study, which began in 2002, could not have been accomplished with animal research [55] .

Similarly, any approach that emphasizes evidence-based prevention would provide benefits to both animals and humans. Resource limitations might require a strategic approach that emphasizes diseases with the greatest public health threats, which increasingly fall within the scope of preventable diseases.

It is clear that there have been many scientific and ethical advances since the first publication of Russell and Burch's book. However, some in the scientific community are beginning to question how well data from animals translates into germane knowledge and treatment of human conditions. Efforts to objectively evaluate the value of animal research for understanding and treating human disease are particularly relevant in the modern era, considering the availability of increasingly sophisticated technologies to address research questions [9] . Ethical objections to the use of animals have been publically voiced for more than a century, well before there was a firm scientific understanding of animal emotion and cognition [15] . Now, a better understanding of animals' capacity for pain and suffering is prompting many to take a closer look at the human use of animals [56] .

Articles in the accompanying Collection only briefly touch on the many scientific and ethical issues surrounding the use of animals in testing and research. While it is important to acknowledge limitations to non-animal methods remain, recent developments demonstrate that these limitations should be viewed as rousing challenges rather than insurmountable obstacles. Although discussion of these issues can be difficult, progress is most likely to occur through an ethically consistent, evidence-based approach. This collection aims to spur further steps forward toward a more coherent ethical framework for scientific advancement.

Acknowledgments

The authors thank the conference speakers and participants for their participation.

Author Contributions

Conceived and designed the experiments: HRF NB. Contributed reagents/materials/analysis tools: HRF NB. Wrote the paper: HRF NB.

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• 16. United States (1947) Nuremberg code. Trials of War Criminals before the Nuremberg Military Tribunals under Control Council Law No. 10. Washington, D.C.: U.S. Government Printing Office. Available: http://ohsr.od.nih.gov/guidelines/nuremberg.html . Accessed 2011 Jan 7.
• 17. World Medical Association (1964) Declaration of Helsinki. 18 th WMA General Assembly. Helsinki, Finland: Available: http://history.nih.gov/research/downloads/helsinki.pdf . Accessed 2011 Jan 7.
• 18. National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research (1979) The Belmont Report. Washington, D.C.: US Department of Health, Education, and Welfare. Available: http://ohsr.od.nih.gov/guidelines/belmont.html . Accessed 2011 Jan 7.
• 19. Parliament of the United Kingdom (1876) Cruelty to Animals Act 1876. Available: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1872363/ . Accessed 2011 Jan 7.
• 20. Animal Welfare Act. 7 U.S.C. §§ 2131–2159.
• 22. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (1996) Guide for the care and use of laboratory animals. Washington, D.C.: National Academy Press. 140 p.
• 23. Office of Laboratory Animal Welfare (2002) Public Health Service policy on humane care and use of laboratory animals. Available: http://grants.nih.gov/grants/olaw/references/phspol.htm#PublicHealthServicePolicyonHumaneCareandUseofLaboratory . Accessed 2011 Jan 18.
• 24. Office of Laboratory Animal Welfare (2002) U.S. Government principles for the utilization and care of vertebrate animals used in testing, research and training. Available: http://grants.nih.gov/grants/olaw/references/phspol.htm . Accessed 2011 Jan 7.
• 25. Gregory NG (2004) Physiology and behavior of animal suffering. Oxford, U.K.: Blackwell Science. 280 p.
• 26. McMillan FD, editor. (2005) Mental Health and Well-Being in Animals. Oxford, U.K.: Blackwell Publishing Professional. 301 p.
• 27. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council (2009) Recognition and alleviation of pain and distress in laboratory animals. Washington, D.C.: National Academy Press. 196 p.
• 31. Panksepp J (2004) Affective neuroscience: the foundations of human and animal emotions. Oxford: Oxford University Press. 480 p.
• 32. Koob GF, Ehlers CL, Kupfers DJ, editors. (1989) Animal Models of Depression. Boston, MA: Birkhäuser. 295 p.
• 39. Shettleworth SJ (1998) Cognition, evolution, and behavior. Oxford, U.K.: Oxford University Press. 704 p.
• 40. deWaal F (2009) The age of empathy: nature's lessons for a kinder society. New York, NY: Random House, Inc. 304 p.
• 44. Burghardt GM (2005) The genesis of animal play: testing the limits. Cambridge, U.K.: MIT Press. 501 p.
• 49. Committee on Toxicity Testing and Assessment of Environmental Agents, National Research Council (2007) Toxicity testing in the 21st century: a vision and a strategy. Washington, DC: National Academy Press. 216 p.
• 55. Dr. Susan Love Research Foundation, National Cancer Institute Cancer Biomedical Informatics Grid (2009) Health of Women Study. Available: http://cabig.cancer.gov/action/collaborations/howstudy/ . Accessed 2011 Jan 10.
• 56. Beauchamp TL, Orlans FB, Dresser R, Morton DB, Gluck JP (2008) The Human Use of Animals: Case Studies in Ethical Choice, 2 nd ed. New York, NY: Oxford University Press. 287 p.

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The Unseen Suffering in Animal Testing

When people hear ‘animal testing,’ some picture harmless scenarios like applying makeup to our furry friends, while others associate it with riskier tests, such as injecting chemicals into animals. However, the reality of animal testing goes beyond these initial impressions. 

Animal experimentation is a procedure performed on living animals to test the safety of consumer goods and to study product development. Many people in the media think animal testing allows scientists to learn more about humans, ensure the safety of treatments for illnesses and products. Although this might sound like it is benefiting us, it is ultimately causing us more harm than good. As a practice often viewed as essential for product safety and medical progress, animal experimentation is commonly misunderstood.

Animals used in experiments can experience psychological distress, including anxiety, depression, and behavioral abnormalities. This is caused by the stress of being confined to small cages, the lack of social interaction, and the fear and uncertainty associated with being used in experiments. Animals used for experiments are merely seen as test subjects, and many are ultimately euthanized either because the experiment is over or because they have become too sick or injured to continue. According to PETA , “Animals in laboratories are treated like disposable laboratory equipment, rather than the thinking, feeling beings they are. Every year, more than 100 million animals are tormented and killed in U.S. laboratories for chemical, drug, food, and cosmetics testing; for medical training; for biology lessons; and for curiosity-driven research.” The emotional and physical pain experienced by laboratory animals, as described by PETA, points out the ethical problem that comes from their use. 

It is clear that scientific research has often fallen short in terms of minimizing pain and suffering in animals. This is primarily because scientists are not legally obligated to do so. Animals subjected to experimentation continue to endure excruciating pain, as there is currently no comprehensive U.S. legislation explicitly prohibiting them from being used in experiments. Although there is the Animal Welfare Act (AWA) , it has many flaws, and animal advocates believe it is not as effective as it should be. The AWA grants experimenters the authority to carry out procedures that involve burning, starvation, decapitation, and other forms of harm to animals.

So, can we get a good result from two different anatomies? Not at all. In one experiment , after receiving a drug injection, human volunteers all experienced a severe, fatal reaction that resulted in organ failure. However, mice, rabbits, and rats who received the same drug by injection showed no negative side effects. Such unreliable research and testing does not result in a human cure or treatment.

Humans and animals differ too much from one another for results to be consistent, reliable, and dependable. Animals absorb, metabolize, and eliminate substances differently than humans do; therefore, animals would react differently to drugs than humans do. Aspirin kills cats and causes birth defects in rats, mice, guinea pigs, dogs, and monkeys. But it simultaneously helps us relieve pain and reduce the risk of serious problems like heart attacks and strokes. This situation is also similar to our perception of chocolate. We see chocolate as a delightful treat, whereas for dogs, consuming chocolate can be life-threatening. 

Reactions to the exposure of these products vary among species, making it difficult to extract data from animal tests and apply them to situations in which humans are exposed. According to the U.S. Food and Drug Administration , 92% of drugs that are shown to be safe and effective in animals fail in human trials. To ensure safer and more successful medicines for humans in the future, alternative techniques should be required.

As many say, up to an extent, it is necessary to experiment on animals for humanity. A Bronx Science student (who wishes to stay anonymous) has stated, “Taking a scientific approach, it is absolutely necessary. For centuries, humans have used rats and various smaller animals to test out chemical, physiological, and psychological effects that can happen to humans. Sure it is unethical, but without animal testing, these ‘research’ and ‘tests’ will be done on humans instead, which is much more unethical.” The argument that animal testing is an unavoidable necessity for scientific research and expansion must be critically examined. While historically it has been used to assess chemical, physiological, and psychological effects to protect humans, it is essential to address the ethical concerns it raises. The assertion that it is unethical but necessary does not sufficiently resolve the moral dilemma at its core. 

Moreover, the assumption that animal testing is the sole reliable method for assessing the effects of substances on humans is increasingly discredited. The suggestion that without animal testing, human experimentation would be the only option is invalid, as there are safer and more effective options. While it is widely acknowledged that conducting tests on humans is ethically problematic, it’s important to note some willingly volunteer for such testing. This approach offers significant advantages in terms of cost-efficiency, time, and accuracy, making it a promising avenue for scientific research and experimentation.

As we delve into the realm of animal welfare and the pursuit of alternatives to animal testing, it is essential to acknowledge the personal perspectives that drive our dedication to this cause. One such perspective is captured by an advocate in AAVS , Nicole Green. Nicole Green is committed to promoting awareness and humane education. She said, “As a person who cares about animals, I feel that animals deserve to live their lives as the unique individuals they are, free from pain and exploitation. This is why I dedicate my life to educating the public on this very important issue.” This heartfelt sentiment shows the profound commitment many individuals share in their tireless efforts to champion the rights and well-being of animals. 

Alternatives to animal testing are increasingly being explored and adopted as more ethical, efficient, and scientifically robust methods to assess the safety and efficacy of products and substances. Yet bigger corporations do not want to use animals because there are no better options as animal experimentation is the “gold standard.” 

One key approach is in vitro testing , which uses human cell cultures and tissue models to mimic biological processes, offering a more accurate representation of human responses. Advanced computer modeling and simulation techniques, known as in silico methods , enable the prediction of toxicological outcomes and drug interactions without actual animal experimentation. Microdosing , in which minimal amounts of a substance are administered to humans to study its effects, is gaining popularity for its potential to replace animal testing in pharmaceutical research. Organ-on-a-chip technology allows the creation of micro-scale systems that replicate the functions of entire organs, providing a platform for testing drug responses and toxicity. 

Collectively, these alternatives not only reduce ethical concerns surrounding animal testing but also offer more precise and human-relevant results, ultimately improving the safety and effectiveness of products and medicines.

The practice of animal testing, often misunderstood and surrounded by ethical dilemmas, raises critical questions about its necessity and implications. In fact, it’s worth noting that current limited legal protections leave countless creatures vulnerable to unending pain, distress, and suffering. Animal testing laws contain such large loopholes that experimenters can get away with just about anything. As advocates for both human and animal welfare, shouldn’t we collectively seek a more compassionate and scientifically stronger path forward?

“As a person who cares about animals, I feel that animals deserve to live their lives as the unique individuals they are, free from pain and exploitation. This is why I dedicate my life to educating the public on this very important issue,” said Nicole Green, an advocate in AAVS.

• advocacy for animal welfare
• alternatives to animal testing
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• animal suffering prevention
• animal testing
• animal testing alternatives
• animal testing opposition
• animal-free research
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November 3, 2022

Controversial monkey study reignites animal testing debate

by Issam AHMED

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, that 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.

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 .

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.

Harm reduction

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 Alzheimers 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 US 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 advisor 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.

Journal information: Proceedings of the National Academy of Sciences

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Ethics of Medical Research with Animals

The Case for Phasing Out Experiments on Primates

Whether they realize it or not, most stakeholders in the debate about using animals for research agree on the common goal of seeking an end to research that causes animals harm. [1]  The central issues in the controversy are about how much effort should be devoted to that goal and when we might reasonably expect to achieve it. Some progress has already been made: The number of animals used for research is about half what it was in the 1970s, and biomedical research has reached the point where we can reasonably begin to envision a time when it could advance without causing harm to animals. With some effort and aggressive development of new biomedical research technologies, full replacement of animals in harmful research is within our grasp. The goal will not be reached all at once, however, and phasing out invasive research on all nonhuman primates should be the priority.

Approximately 70,000 nonhuman primates are used for research in the United States each year, according to the U.S. Department of Agriculture, and another 45,000 are held or bred for research. They include macaques, baboons, marmosets, and other monkeys, as well as some chimpanzees. Moreover, these numbers are increasing in the United States and Canada. The rise is driven in part by the “high-fidelity” notion (supported by very little careful scientific justification) that primates are likely to be better models than mice and rats for studying human diseases, and partly by the sheer availability of primates.

The availability factor is a result of historical accident. In the 1960s, the United States invested in a significant infrastructure for primate research through creation of the National Primate Research Centers. The primate center program was the result of two unrelated occurrences. First, in the 1950s, hundreds of thousands of wild primates were captured and imported to support the race to develop a poliomyelitis vaccine. By 1960, with polio vaccines in use, this “race” was essentially over, but laboratories still had tens of thousands of primates. Then, they became swept up in another kind of race. The Russians had beaten the United States into space by launching the first satellite, creating panic that Russian science was outpacing U.S. science. American scientists made the argument that, because the Russians had a big primate research center, the United States should also have one or more primate centers. Seven facilities, formally recognized as government-supported institutions, were set up to provide support for and opportunities to do research in nonhuman primates.

The centers did not produce the hoped-for results. Three federal assessments found that the research conducted by the centers fell far short of expectations in terms of quality, and many deficiencies were also noted. [2]  In the early 1980s, these centers were “rescued,” in a sense, by the discovery that primates at the California Regional Primate Research Center were suffering from a simian version of AIDS. Suddenly, there was renewed focus on research in nonhuman primates. There are now eight National Primate Research Centers, the objective of which continues to be “to provide support for scientists who use NHPs in their research.” [3]

Primates are used for a wide variety of research purposes. An analysis of one thousand federally funded studies that involved nonhuman primates found that research on HIV accounted for about 27 percent of the funding, followed by colony maintenance (likely because caring for primates is costly) at 15 percent, neurological research at 14 percent, and developmental research at 10 percent. [4]

Arguments for Phasing Out Primate Research

Phasing out primate use should be a priority for ethical, scientific, and economic reasons. The ethical concerns fall into two categories. One of them is the nature of the primates themselves. They are well known for their cognitive and emotional abilities. Studies demonstrate that they have mathematical, memory, and problem-solving skills and that they experience emotions similar to those of humans—for example, depression, anxiety, and joy. Chimpanzees can learn human languages, such as American Sign Language. Primates also have very long lifespans, which is an ethical issue because they are typically held in laboratories for decades and experimented on repeatedly.  The other category of ethical concern is how primates are treated. Each year, thousands are captured from the wild, mostly in Asia and Mauritius, and transported to other countries. For example, China sets up breeding colonies, and the infants are sold to various countries, including the United States and European countries.  The animals experience considerable stress, such as days of transport in small crates and restrictions on food and water intake. Studies show that it takes months for their physiological systems to return to baseline levels, [5]  and then they face the trauma of research, including infection with virulent diseases, social isolation, food and water deprivation, withdrawal from drugs, and repeated surgeries.

Providing for the welfare of primates in a laboratory setting is very challenging. According to the Animal Welfare Act, each facility must develop and follow a plan for environmental enhancement to promote the psychological well-being of nonhuman primates. The plan must address social grouping; enriching the environment, with special consideration for great apes; caring for infants, young juveniles, and those primates showing signs of psychological distress; and ensuring the well-being of those primates who are used in a protocol requiring restricted activity.

Social companionship is the most important psychological factor for most primates. Federal law requires institutions to house primates in groups unless there is justification, such as debilitation as a result of age or other conditions, for housing them alone. But a recent analysis of documents from two large facilities obtained by The Humane Society of the United States demonstrates that primates spent an average of 53 percent of their lives housed alone. In many instances, a metal shape hung for a month on the bars of a metal cage was deemed to constitute adequate “enrichment.” [6]

As we have done with chimpanzees, we need to critically analyze uses of other nonhuman primates. A good starting point would be the formation of a working group  of diverse stakeholders who agree that ending primate research is a worthwhile goal.

The Bateson report recommended that all proposed primate studies be assessed using the following parameters: scientific value, probability of medical or other benefit, availability of alternatives, and likelihood and extent of animal suffering. [9]   The report indicated that if a proposed use would cause severe suffering, it should be allowed only if there is a high likelihood of benefit. The report considered approximately 9 percent of the studies it examined to be of low importance and to inflict high levels of suffering. [10]  The report was critical of some of the neuroscience research, which represented nearly half of the research surveyed. It found that half of the thirty-one neuroscience studies took a high toll on animal welfare, although most were also considered to be of high scientific value. Two of the studies were of concern because they posed a “high welfare impact,” but moderate-quality science and little medical benefit. [11]  The report recommended that more consideration be given to alternatives to nonhuman primates, including brain imaging, noninvasive electrophysiological technologies, in vitro and in silico techniques, and even research on human subjects. [12]  The report recommended other ways of reducing the number of primates needed for research, including data sharing, publication of all results, and periodic review of outcomes, benefits, and impact of the research. “Researchers using NHPs have a moral obligation to publish results—even if negative—in order to prevent work from being repeated unnecessarily,” the report states. [13]

In addition to the ethical and scientific arguments for ending research involving primates, there are economic reasons. Primates are very expensive to maintain. The eight National Primate Research Centers alone receive $1 billion of the National Institutes of Health’s total $32 billion budget. The care and upkeep of primates other than chimpanzees is twenty to twenty-five dollars per day, compared with twenty cents to about $1.60 per day for small rodents. We argue that much of the research with nonhuman primates is either of questionable value or has not been carefully evaluated and justified. Therefore, these funds might be better spent on other research models, including several technologies that could replace nonhuman primates and other animals. Francis Collins, director of the NIH, argued in 2011 that new high-throughput approaches could overcome the drawbacks of animal models—they are slow, expensive, and not sufficiently relevant to human biology and pharmacology. [14]

Several such technologies are available. The U.S. Army recently announced that it would end the use of monkeys for chemical casualty training courses and replace them with alternatives such as simulators that mimic the effects of nerve gas on victims. [15]

Following Chimpanzees

The process that culminated in the phasing out of invasive research on chimpanzees in the United States in 2011 can and should be applied to all other nonhuman primates. Public opinion and ethical challenges drove that process. Even before the 2011 IOM report, scientists in the United States were having difficulty justifying why they should perform experiments on chimpanzees when their colleagues in other countries had stopped doing so. Unlike nonhuman primates in general, the number of chimpanzees in U.S. labs has been declining since reaching its peak in the late 1990s.

The main drivers for efforts to phase out research on chimpanzees are their genetic, biological, and behavioral similarities with humans. [16]  Chimpanzees are humans’ closest relative. Chimpanzee cognition has been studied extensively, and their capabilities are considerable. As with other primates, the impact of laboratory life—including barren housing and social isolation—on chimpanzees can last decades due to their long lifespan and thus raises significant welfare concerns. There is evidence that some chimpanzees used in research suffer from a form of posttraumatic stress disorder similar to that of humans. In their 2008 article, Gay Bradshaw and colleagues described the plight of a chimpanzee named Jeannie who endured invasive research and social isolation for over a decade. She exhibited abnormal behavior, including self-injury, bouts of aggression, and, according to laboratory documentation, a “nervous breakdown.” When retired to a sanctuary, she recovered partially, but was ultimately diagnosed with complex PTSD. The paper concluded: “The costs of laboratory-caused trauma are immeasurable in their life-long psychological impact on, and consequent suffering of, chimpanzees.” [17]

As we have done with chimpanzees, we need to critically analyze current uses of other nonhuman primates, the viability of alternative models, and the economic issues involved to forge the best way forward. A good starting point would be the formation of a working group of diverse stakeholders who agree that ending primate research is a worthwhile goal. Such a working group—possibly organized by the NIH and the National Academies—would analyze the necessity of primate use and identify existing and potential alternatives.

The stakeholder group could develop a concrete plan to work on common-ground issues. This would involve developing priorities, short-term outcomes, and related activities. The ongoing Human Toxicology Project Consortium’s work to ultimately replace all animals for toxicity testing is a good example of this approach. (See “No Animals Harmed:  Toward a Paradigm Shift in Toxicity Testing,” in this volume.) The mission of the consortium is to “serve as a catalyst for the prompt, global, and coordinated implementation of ‘21 st Century’ toxicology, which will better safeguard human health and hasten the replacement of animal use in toxicology.” [18]   Because science is ever-changing, there must be an ongoing analysis of new technologies and challenges, and regulatory authorities must adjust regulations accordingly. In the United States, many stakeholders express frustration with the fact that the Food and Drug Administration, for example, favors data from outdated tests, including those that involve primates and other animals.

Phasing out invasive research on all nonhuman primates would take courage on the part of leaders in science and policy. It is a formidable task, but similarly transformative changes in how we conduct biomedical research have been achieved. At various points in the past century and a quarter, restrictions have been placed on particular kinds of human and animal research because of ethical issues, despite objections that such restrictions would slow scientific progress; think, for instance, of the Helsinki Declaration to protect human subjects in research and the animal welfare laws in the United States and the European Union. However, these laws have not slowed the pace of discovery about biology and disease processes. If anything, there has been an acceleration of such discovery in the half-century since these restrictions went into effect.

In the early 1950s, Sir Peter Medawar pressed the Universities Federation for Animal Welfare to develop a report on how laboratory animal welfare could be improved and how distress and suffering in the research laboratory might be reduced. That initiative led to publication of a volume on humane experimental approach that is now regarded as the foundation for the concept of the Three Rs of replacement, reduction, and refinement of animal studies. [19]  Ten years later, in 1969, Medawar correctly predicted that laboratory animal use would peak within ten years and then start to decline. He argued that animal research would allow researchers to develop the knowledge and understanding that would lead, eventually, to the replacement of animal use in laboratories. In 2010, forty years after Medawar’s prediction, laboratory animal use is approximately 50 percent of what it was in 1970. Francis Collins has pointed to the down sides of animal-based research—that is “time-consuming, costly, and may not accurately predict efficacy in humans.” [20]   He has also suggested that nonanimal technologies might be quicker and more effective in new drug discovery programs. Given the trends and political will, we believe that we could reach Medawar’s prediction of complete replacement by 2050.

Now is the time for an internationally coordinated effort to define a strategy to replace all invasive research on primates. At the very least, we need to move quickly to reverse the increase in laboratory primate use in the United States and Canada. Until replacement is a realistic option, we must reduce the number of primates used and refine studies to reduce their suffering, for the sake of both animal welfare and science.

Kathleen M. Conlee is vice president for animal research issues with The Humane Society of the United States. She worked for several years at a primate breeding and research facility and also worked with great apes in a sanctuary setting. Her current work focuses on the long-term goal of replacing the use of animals in harmful research and testing, the ongoing development of nonanimal alternatives, and the short-term goals of ending invasive chimpanzee research and retiring chimpanzees from laboratories to sanctuaries. 

Andrew N. Rowan is chief scientific officer at The Humane Society of the United States and chief executive officer of The Humane Society International. He has written numerous books and peer-reviewed publications regarding animal research, including a book titled The Animal Research Controversy: Protest, Process and Public Policy (Center for Animals and Public Policy, Tufts University School of Veterinary Medicine, 1995). He is a biochemist in training, and a focus of his career has been promotion of the three Rs in animal research: replacement of nonhuman animals, reduction in number of animals used, and refinement to decrease animal suffering.

[[12]]12. Ibid., 4, 5, 16.[[12]

• 1. C. Blakemore, “Should We Experiment on Animals? Yes,” Telegraph, October 28, 2008. ↵
• 2. A.N. Rowan, Of Mice, Models and Men (Albany: State University of New York Press, 1984). ↵
• 3. Department of Health and Human Services, Funding Opportunity for the National Primate Research Centers, http://grants.nih.gov/grants/guide/pa-files/PAR-11-136.html, accessed July 7, 2011. ↵
• 4. K.M. Conlee, E.H. Hoffeld, and M.L. Stephens, “A Demographic Analysis of Primate Research in the United States,” Alternatives to Laboratory Animals 32, suppl. 1A (2004): 315-22. ↵
• 5. P.E. Honess, P.J. Johnson, and S.E. Wolfensohn, “A Study of Behavioural Responses of Non-Human Primates to Air Transport and Re-Housing,” Laboratory Animals 38,  no. 2 (2004): 119-32; J.M. Kagira et al., “Hematological Changes in Vervet Monkeys ( Chlorocedub aethiops ) during Eight Months’ Adaptation to Captivity,” American Journal of Primatology 69, no. 9 (2007): 1053-63. ↵
• 6. J. Balcombe and K.M. Conlee, “Primate Life in Two American Laboratories,” presentation to the Eighth World Congress on Alternatives and Animal Use in the Life Sciences, held in Montreal, Quebec, Canada, on August 21-25, 2011. ↵
• 7. P. Bateson, A Review of Research Using Nonhuman Primates: A Report of a Panel Chaired by Professor Sir Patrick Bateson, FRS (London and Wiltshire, U.K.: Biotechnology and Biological Sciences Research Council, Medical Research Council, and Wellcome Trust, 2011) http://www.mrc.ac.uk/Utilities/Documentrecord/index.htm?d =MRC008083; J.A. Smith and K.M. Boyd, eds., The Use of Non-Human Primates in Research and Testing (Leicester, U.K.: British Psychological Society, 2002); D. Weatherall, The Use of Non-Human Primates in Research: A Working Group Report Chaired by Sir David Weatherall FRS FmedSci (London: Academy of Medical Sciences, 2006), http://www.acmedsci.ac.uk/images/project/nhpdownl.pdf. ↵
• 8. Institute of Medicine, Committee on the Use of Chimpanzees in Biomedical and Behavioral Research, Chimpanzees in Biomedical and Behavioral Research: Assessing the Necessity (Washington, D.C.: National Academies Press, 2011), 4. ↵
• 9. Bateson, A Review of Research Using Nonhuman Primates , 2. ↵
• 10. Ibid., 1. ↵
• 11. Ibid., 12-13. ↵
• 13. Ibid., 3. ↵
• 14. F.S. Collins, “Reengineering Translational Science: The Time Is Right,” Science Translational Medicine 3, no. (2011): 1-6. ↵
• 15. B. Vastag, “Army to Phase Out Animal Nerve-Agent Testing,” Washington Post, October 13, 2011. ↵
• 16. G.W. Bradshaw et al., “Building Inner Sanctuary: Complex PTSD in Chimpanzees,” Journal of Trauma and Dissociation 9, no. 1 (2008): 9-34; J.A. Smith and K.M. Boyd, eds., The Boyd Group Papers on the Use of Non-Human Primates in Research and Testing (Leicester, U.K.: British Psychological Society, 2002). ↵
• 17. Bradshaw et al., “Building Inner Sanctuary,” 31. ↵
• 18. Human Toxicology Project Consortium Web site, http://htpconsortium.wordpress.com/about-2, accessed February 13, 2012. ↵
• 19. W.M.S. Russell and R.L. Burch, The Principles of Humane Experimental Technique (London: Methuen, 1959). ↵
• 20. F.S. Collins, “Reengineering Translational Science,” 3. ↵

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FBR: The case for animal testing

• Thought Leaders
• June 19, 2017

Thanks to research with animals, in particular dogs, our pets are able to live healthier and longer lives than they ever have before. Every vaccine developed for your pet was developed with the help of animals. And the fact that pet owners are able to give their dogs and cats the best possible nutrition available, is owed to the fact that other dogs and cats have served in research. Animal testing is a controversial topic and individuals and organisations associated with this type of research have become targets of illegal actions by animal rights activists. What is the impact of this hostility? Before the Animal Enterprise Terrorism Act was passed by Congress and signed by the President in 2006, FBR tracked approximately 40 illegal incidents per year committed against researchers and institutions. After the law went into effect, the number of illegal incidents in the US has dropped to virtually zero. The impact is very clear. Aside from the immense fear for the personal safety of researchers and their families, these actions contributed to, in some cases, the loss of decades-worth of scientific data, and significant delays in the ability to combat illness and cure disease. These illegal actions unequivocally presented a material risk to universities, pharmaceutical companies and the individuals who worked for them. What is your approach to dealing with it? Though illegal incidents have declined precipitously, tactics by opponents of animal research have changed. Today, animal rights activists spend more time applying pressure through social media channels, traditional media, and public demonstrations designed to shock their audience. They do these things in the hope of generating the attention of elected officials in order to ultimately restrict or eliminate research with animals. What do animal experiments tell us about humans? Because of the genetic similarities of animals and people, animals are invaluable models for human disease and are critical in the studies of incurable diseases like Zika, Ebola, Alzheimer’s, Parkinson’s, HIV/AIDS, and Malaria. Without research animals, the world may never have seen the deployment of vaccines against typhus, yellow fever, and polio.

To give you an idea of the genetic similarities of animals and people, here are some facts and figures:

• Of the approximately 4000 genes studied, less than 10 are found in one species and not the other.
• On average, the protein-coding regions of the mouse and human genomes are 85 per cent identical; some genes are 99 per cent identical, while others are only 60 per cent identical. These regions are evolutionarily conserved because they are required for function (https://www.genome.gov/10001345/).
• Monkeys and humans share anywhere between 96 per cent to 99 per cent of genes.
• Scientists have sequenced the genome of the chimpanzee and found that humans are 96 per cent similar to the great ape species (NatGeo).
• Humans and chimps share a surprising 98.8 per cent of their DNA (American Museum of Natural History).

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Facts about FDA and Animal Welfare, Testing & Research 

Medical and veterinary products save lives every day in the U.S. FDA-regulated products like blood pressure medicine, chemotherapy, and MRI machines help people and animals live longer and healthier. The FDA regulates human and animal medical products to ensure they are safe and effective. 

Products undergo different types of testing to determine their safety and effectiveness. These tests may include animal testing, and almost always include other types of tests. Here are some facts about animal testing of FDA-regulated medical products and alternatives to animal testing.     

Fact: The FDA encourages and accepts scientifically valid alternatives to animal testing. However, validated alternatives to animal testing are not available yet for many medical products.

• Laboratory tests in a petri dish or test tube, which may include tests with human or animal cells and tissues. 
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• Animal testing. 

Fact: The FDA supports the development of alternatives to animal testing.  

Fact: federal laws regulate the treatment of test or research animals. .

• The Animal Welfare Act and Animal Welfare Act Regulations from the U.S. Department of Agriculture (USDA).
• The Public Health Service Policy of Humane Care and Use of Laboratory Animals from the National Institutes of Health Office of Laboratory Animal Welfare (OLAW).
• The Guide for the Care and Use of Laboratory Animals from the National Research Council.

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The ethics of animal experimentation.

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

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

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

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

• The Case Against Animal Experimentation
• The Case For Animal Experimentation
• A Middle Ground

The Case Against Animal Experimentation ^

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

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

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

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

The Case For Animal Experimentation ^

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

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

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

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

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

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

A Middle Ground ^

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

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

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

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

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

For further reading:

• Singer, Peter. “All Animals are Equal.” Ethics in Practice . LaFollette, Hugh ed. Blackwell Publishing. 2007. Peter Singer is one of the best publicly known advocates of animal rights and animal equality. This philosophical essay briefly presents his views.
• Fox, Michael Allen. “The Moral Community.” Ethics in Practice. LaFollette, Hugh ed. Blackwell Publishing. 2007. This essay defends animal experimentation.
• Frey, R.G. “Animals and Their Medical Use.” Contemporary Debates in Applied Ethics. Cohen, Andrew and Wellman, Christopher eds. Blackwell Publishing. 2005 In this essay Frey puts forth a view where animals do matter, but human welfare is considered more important.
• Regan, Tom. “Empty Cages: Animals Rights and Vivisection.” Contemporary Debates in Applied Ethics. Cohen, Andrew and Wellman, Christopher eds. Blackwell Publishing. 2005. This essay supports animal rights.
• “Ethics and Alternatives”. Research Animal Resources. University of Minnesota. 2003. Ethics and Alternatives for Animal Use in Research and Teaching . A great resource describing some ways to minimize the use of animals in research and to practice the best standards when using animals.

– Adam Hepworth, 11-26-08

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HOPES is a team of faculty and undergraduate students at Stanford University dedicated to making scientific information about Huntington’s disease (HD) more readily accessible to the public. Our goal is to survey the rapidly growing scientific literature on HD and to present this information in a web source.

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EPA Leads International Case Study to Reduce Animal Testing for Chemical Safety

Published November 4, 2019

EPA is prioritizing efforts to reduce, refine, and replace animal testing in chemical safety research. In a new case study recently published in Toxicological Sciences  , EPA identified an innovative approach to rapidly screen chemicals for biological impacts using new “ in vitro ,” or “in test-tube” methods.  This work supports the September 2019 EPA Administrator Memo advocating the benefits of new approach methods for predicting potential hazards without the use of traditional methods that rely on animal testing.

Researches led by EPA’s Katie Paul Friedman used high throughput toxicological methods to explore the scale at which in vitro techniques could be used as an alternative to current animal-based techniques for calculating a chemical’s POD. High throughput in vitro experiments use automated technologies ( including robots! ) to expose living cells or proteins to chemicals. The cells or proteins are then screened for changes in biological activity that may suggest toxic effects. Based on the dose at which the potential toxic effects are observed, scientists calculate a POD value that is meant to model how different types of cells might respond to chemicals if they were in the human body.

Although modeling the interactions of chemicals in vitro is challenging and may not fully represent what occurs inside a living human body, in vitro methods present several advantages over traditional animal-based testing. First, in vitro studies require fewer resources, which saves time, cost, and animals.

“These savings enable the screening of many more chemicals, providing more information to decision makers about which chemicals may be more interesting for investment of additional resources,” says Katie.

The team found that for 90% of nearly 500 chemicals reviewed, the in vitro -based methods provide a POD value less than or equal to the animal-derived POD values. These results gave scientists confidence that the use of in vitro -based POD doses may provide an alternative that is equivalent to current techniques.

To support this research, EPA is working with international scientists from regulatory agencies in Canada, the European Union, and Singapore as part of a global workshop known as “Accelerating the Pace of Chemical Risk Assessment” (APCRA). EPA and APCRA will continue meeting annually to discuss the results of this and other case studies exploring in vitro risk assessment techniques. For more information about how EPA and its partners are changing the game in chemical safety, read about our computational toxicology and exposure research.

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Virome Sequencing Identifies H5N1 Avian Influenza in Wastewater from Nine Cities

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Avian influenza (serotype H5N1) is a highly pathogenic virus that emerged in domestic waterfowl in 1996. Over the past decade, zoonotic transmission to mammals, including humans, has been reported. Although human to human transmission is rare, infection has been fatal in nearly half of patients who have contracted the virus in past outbreaks. The increasing presence of the virus in domesticated animals raises substantial concerns that viral adaptation to immunologically naïve humans may result in the next flu pandemic. Wastewater-based epidemiology (WBE) to track viruses was historically used to track polio and has recently been implemented for SARS-CoV2 monitoring during the COVID-19 pandemic. Here, using an agnostic, hybrid-capture sequencing approach, we report the detection of H5N1 in wastewater in nine Texas cities, with a total catchment area population in the millions, over a two-month period from March 4 th to April 25 th , 2024. Sequencing reads uniquely aligning to H5N1 covered all eight genome segments, with best alignments to clade 2.3.4.4b. Notably, 19 of 23 monitored sites had at least one detection event, and the H5N1 serotype became dominant over seasonal influenza over time. A variant analysis suggests avian or bovine origin but other potential sources, especially humans, could not be excluded. We report the value of wastewater sequencing to track avian influenza.

Competing Interest Statement

The authors have declared no competing interest.

Funding Statement

This work was supported by S.B. 1780, 87th Legislature, 2021 Reg. Sess. (Texas 2021) (E.B., A.W.M., and J.F.P.), NIH/NIAID (Grant number U19 AI44297) (A.W.M.), Baylor College of Medicine Melnick Seed (A.W.M) and Alkek Foundation Seed (J.F.P.), and Pandemic Threat Technology Center (P.A.P.).

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I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Data Availability

All data produced are available online at https://zenodo.org/doi/10.5281/zenodo.11175923 and NCBI SRA BioProject: PRJNA966185

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Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows

• Sai Zhang 1 , 2 ,
• Juan C. Marini 3 ,
• Vengai Mavangira 4 ,
• Andrew Claude 4 ,
• Julie Moore 1 ,
• Mahmoud A. Mohammad 3 &
• Nathalie L. Trottier 1 , 5  

Journal of Animal Science and Biotechnology volume  15 , Article number:  65 ( 2024 ) Cite this article

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Metrics details

The study objective was to test the hypothesis that low crude protein (CP) diet with crystalline amino acids (CAA) supplementation improves Lys utilization efficiency for milk production and reduces protein turnover and muscle protein breakdown. Eighteen lactating multiparous Yorkshire sows were allotted to 1 of 2 isocaloric diets (10.80 MJ/kg net energy): control (CON; 19.24% CP) and reduced CP with “optimal” AA profile (OPT; 14.00% CP). Sow body weight and backfat were recorded on d 1 and 21 of lactation and piglets were weighed on d 1, 14, 18, and 21 of lactation. Between d 14 and 18, a subset of 9 sows (CON = 4, OPT = 5) was infused with a mixed solution of 3-[methyl- 2 H 3 ]histidine (bolus injection) and [ 13 C]bicarbonate (priming dose) first, then a constant 2-h [ 13 C]bicarbonate infusion followed by a 6-h primed constant [1- 13 C]lysine infusion. Serial blood and milk sampling were performed to determine plasma and milk Lys enrichment, Lys oxidation rate, whole body protein turnover, and muscle protein breakdown.

Over the 21-d lactation period, compared to CON, sows fed OPT had greater litter growth rate ( P < 0.05). Compared to CON, sows fed OPT had greater efficiency of Lys ( P < 0.05), Lys mammary flux ( P < 0.01) and whole-body protein turnover efficiency ( P < 0.05). Compared to CON, sows fed OPT tended to have lower whole body protein breakdown rate ( P = 0.069). Muscle protein breakdown rate did not differ between OPT and CON ( P = 0.197).

Feeding an improved AA balance diet increased efficiency of Lys and reduced whole-body protein turnover and protein breakdown. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

The increasing availability of crystalline amino acid (CAA) at competitive costs relative to protein ingredients allows for reduction of excessive dietary nitrogen (N) and improving AA balance [ 1 ]. Several studies have shown that improving dietary AA balance in lactating sows leads to greater milk casein yield [ 2 , 3 ] and utilization efficiency of N and essential amino acid (EAA) [ 4 , 5 ] while dramatically mitigating N losses and ammonia emissions to the environment [ 2 ]. Lysine efficiency values previously reported [ 4 , 5 , 6 ] were estimated based on lean mass change during lactation. This approach yielded similar efficiency estimates for Val based on isotopic method [ 7 ]. Lysine utilization efficiency values in lactation using an isotopic approach have not been reported.

The increased apparent AA efficiency may be at the expense of sow body weight (BW) loss and reduced maternal N retention whereby partitioning of dietary AA and energy towards the mammary glands appears to be favoured [ 3 , 5 , 8 ]. Preserving maternal N pool during the lactation period is important since maternal body protein and lipid loss can affect subsequent production performance. Loss in performances may include delayed estrus [ 9 ], reduction of piglet birth weight and litter uniformity [ 10 , 11 ], and prolonged interval from weaning to successful pregnancy [ 12 ], thus compromising the overall life span production efficiency. It is unknown whether the reduced maternal N retention previously reported [ 3 , 5 ] in sows fed an improved AA balance diet was a result of greater maternal body protein breakdown. In addition, whole body and muscle protein breakdown rates in lactating sows are unknown and such values are critical to assess at a mechanistic level the impact of improved dietary AA balance on body protein dynamic. Isotope technique allows for better mechanistic understanding of protein dynamics, including protein turnover rate, AA flux and muscle protein breakdown in humans [ 13 ] and animals [ 14 ].

We hypothesized that low CP diet with improved AA balance would increase milk yield through improving efficiency of Lys for milk and increasing maternal body and muscle protein breakdown. The objectives were to (1) measure whole body protein dynamics and (2) estimate Lys utilization efficiency for milk synthesis.

Materials and methods

Dietary treatments.

The NRC model [ 6 ] was used to estimate requirements for AA, net energy (NE), calcium (Ca) and phosphorus (P). The requirements were predicted based on the following parameters: sow BW of 210 kg after parturition, parity number of 2 and above, average daily intake of 6 kg/d, litter size of 10, piglet average daily gain (ADG) of 280 g/d over a 21-d lactation period, and an ambient temperature of 20 °C. The model predicted a minimum sow BW loss of 7.5 kg and the protein to lipid ratio in the model was adjusted to the minimum allowable value of near zero. The model predicted SID Lys requirement of 0.90% and NE requirement of 2,580 kcal/kg.

A control diet (CON) was formulated using corn and soybean meal as the only sources of Lys to meet the SID Lys requirement (0.90%) and consequently contained 19.24% CP and a SID Val concentration 0.77% which was near the NRC (2012) requirement of 0.79%. All other EAA SID contents were in excess relative to NRC (2012). The SID AA values of feed ingredients were referred to NRC (2012). A second diet was formulated to improve AA balance [ 5 ], and referred to as optimal diet (OPT) throughout the manuscript. Fermentable fiber was high in CON due to high content of soybean meal with 24.88% fermentable fiber [ 6 ]. Thus, the same fiber source (soy hulls) was supplemented in OPT, and levels of fermentable fiber were consistent between CON and OPT. Ingredients and calculated nutrient composition of CON and OPT diets are presented in Table 1 . Analyzed total (hydrolysate) and free AA concentrations are presented in Table 2 , in order to verify the precision of diet formulation. The analyzed N concentration corresponded to a CP% of 18.44 compared to a calculated value of 19.24% CP. Therefore, the analyzed CP concentration value is used in the heading for the remainder of tables.

Animals and feeding

The study was conducted at the Michigan State University Swine Teaching and Research Center. A total of 18 purebred multiparous (parity 2+) Yorkshire sows were moved to conventional farrowing crates between d 105 and 107 of gestation, grouped by parity, and randomly assigned to 1 of 2 dietary treatments within parity groups (CON, n = 9; OPT, n = 9). The study was conducted over 3 blocks of time, with 6, 6, and 5 sows in each block, respectively. One sow in CON from block 3 was removed due to poor feed intake that was deemed unrelated to the dietary treatments. Sows were adapted to the experimental diets (2.2 kg/d) 4 to 6 d before the expected farrowing date. Following farrowing, sows feed allowance progressively increased from 1.88 kg/d on d 1 to 7.44 kg/d at d 21, according to the NRC model [ 6 ], with a targeted ADFI of 6.0 kg/d over the 21-d lactation period. Feed was provided daily in 3 equal meals (0700, 1300, and 1900) with feed intake and refusal recorded daily before the morning meal. On infusion days (between d 14 and 18), the 0700 and 1300 meals were divided into 6 aliquots fed every 2 h from 0700 to 1700. Water was freely accessible to sows and piglets. Litters were aimed to be standardized to 11 piglets within the first 24 h after farrowing with the objective of weaning 10 piglets per sow. Injection of iron and surgical castration of male were conducted on d 1 and 7, respectively, according to the institutional research farm protocol. No creep feed was supplied to the piglets. Body weight and backfat thickness [ 5 ] of sows were recorded on d 1 and 21, and litter weights were recorded on d 1, 14, 18 and 21. Milk yield was estimated for peak lactation (between d 14 and 18) [ 5 ]. Prediction equation for milk yield during peak lactation is as follows [ 15 ]: \(\mathrm{Daily}\;\mathrm{milk}\;\mathrm{yield}\;(\text{g}/\text{d})=\mathrm{littersize}\times(582+1.168\times\text{ADG}+0.00425\times\text{ADG}^2)\)

Bilateral ear vein catheterization

A subset of 10 sows (5 sows per treatment) was used for the catheterization and infusion protocol. An ear vein catheter was placed in each ear, with one ear serving as the infusion line and the other as the sampling line. For the length of the catheterization procedure, piglets were removed and transferred to an empty adjacent stall with a heat lamp. The sows were restrained with a rope snare and remained in their farrowing stall where sedation was induced. For sedation, Telazol was reconstituted with 2.5 mL of 100 mg/mL ketamine and 2.5 mL of 100 mg/mL xylazine to a volume of 5 mL. This sedative mixture was administered i.m. in the brachiocephalicus muscle approximately 6 cm caudal to the ear, at a dosage of 0.1 mL/4.537 kg body weight. Sows were carefully assisted to facilitate laying in ventral recumbence. Sedation lasted for 45 to 60 min. The depth of anesthesia was monitored by the degree of muscle relaxation and respiration rate (i.e., 10 to 25 breaths/min).

The entire dorsal surface of both ears was prepared for aseptic placement of the ear vein catheters. The skin was scrubbed gently with 10% betadine solution followed with 70% isopropyl alcohol. The hair covering the skin area caudal to the ear and dorsal to the neck was clipped using a professional clipper to ensure a good adhesion of veterinary adhesive tape to the skin (described below).

A pre-cut 61-cm, round tip, medical grade microbore intravascular tubing (1.65 mm o.d., 1.02 mm i.d.) with hydromer coating (Access Technology Corp., Skokie, IL, USA) was prefilled at the time of catheterization with heparinized saline (30 IU/mL) before insertion. A hand tourniquet was applied at the base of the ear to distend the medial and lateral branches of the auricular vein. Either vein was used for catheterization. A short-term stylet catheter (14G, 5.08 cm, Safety IV catheter; B. Braun Melsungen AG, Germany) was inserted into the vein with the needle bevel facing up. Upon appearance of blood, the vein was gently occluded, and the needle rotated 180° to angle the bevel facing down. While holding the needle in place, the stylet catheter was gently pushed into the vein through the needle. Once the stylet was in place, the needle was removed, and the intravascular tubing was inserted through the stylet and pushed for approximately 30 cm caudally to reach the external jugular vein, and the catheter verified for patency at this point. Small sections of tape (5.1 cm wide, ZONAS® porous tape, Johnson & Johnson Consumer Companies, Inc., Skillman, NJ, USA) were affixed to the remaining section of intravascular tubing and sutured to the skin to secure the tubing in place. The stylet catheter was also sutured (Monocryl, CP-1, 36 mm, 1/2c; Ethicon Inc., USA) to the skin at the point of entry. Gauze was placed over each sutured sites and held in position by wrapping the ear with elastic adhesive tape. A connector was used to join the intravascular tubing to a long tubing extension (approximately 120 cm). A blunt-end needle adapter with an adaptor injection cap and a male luer lock was placed onto the distal end of the tubing extension. The same vein catheterization procedure was done on the other ear. A final layer of elastic adhesive tape (7.5 cm wide, 3M veterinary adhesive tape) was used to wrap each ear into a gently folded cone shape and to affix extension tubing directly onto the clipped skin surface. The extension tubing ran from the ears to the dorsal region of the neck, caudally to the ears and cranial to the shoulders and the free end (approximately 100 cm) rolled up and placed in a handmade denim protective pouch mounted on 4.0-cm thick foam material. The pouch was kept in place by gluing the foam directly onto the skin with Livestock ID Tag Cement (W.J. Ruscoe Company, Akron, OH, USA). Catheters were verified for patency once more and the lines were filled with sterile saline, coiled, and placed in the pouch until used for infusion and blood sampling. The entire procedure was done following sterile techniques and lasted 45 to 90 min per sow. As soon as sows were able to stand, 15-cm wide elastic bandage (Novation ® , Hartmann USA, Inc., Rock Hill, SC, USA) was wrapped over the pouch and around the neck and thorax in at least 3 layers in the shape of a life vest (crisscross) to protect the pouch. Thereafter, the catheters were verified for patency and flushed with sterilized heparinized saline (30 IU/mL) twice per day.

Catheters were removed after all infusions and blood sampling were completed (blood sampling lasted for 3 d for 3MH; Fig. 1 ). The elastic bandaging was removed, and the elastic adhesive tape was carefully pulled to expose the sutures. The sutures were cut with small surgical scissors, the catheters were gently pulled out of the ear veins, and pressure was applied over the insertion sites to accelerate coagulation. The remaining adhesive tape was then carefully removed, and the pouch was freed from the foam which remained on the sow. Rectal temperature was recorded from the day of catheterization and for 3 d following removal of catheters.

Plasma isotopic enrichment of 3-[methyl- 2 H 3 ]histidine following 3-[methyl- 2 H 3 ]histidine bolus infusion during peak lactation (between d 14 and 18) for sows fed control (CON; 18.4% CP; n = 4) and optimal (OPT; 14.0% CP; n = 4) diets. Plasma isotopic enrichment of 3-[methyl- 2 H 3 ]histidine differed between diets ( P < 0.001) and time points ( P < 0.001), with no interaction between diet and time ( P = 0.894). Standard error of the mean (SEM) = 0.214

Preparation of isotope solutions

Tracers were weighed, dissolved in saline and the solution sterilized by filtration through Millipore Steriflip filters (0.22 μm). For each sow, the following stock solutions were prepared: 3-[methyl- 2 H 3 ]histidine (183 μmol in 20 mL saline for bolus injection), [ 13 C]bicarbonate (368 μmol in 20 mL saline for prime and 736 μmol in 30 mL saline for 2-h infusion), and [1- 13 C]lysine (1.28 mmol in 30 mL saline for prime and 9.00 mmol in 60 mL saline for 6-h infusion). The bolus dose of 3-[methyl- 2 H 3 ]histidine (3MH) was calculated based on 20% pool size of 3MH in sows [ 16 , 17 ]. The infusion rate of [1- 13 C]lysine was calculated based on average flux of lysine (25 mmol/h) in lactating sows [ 7 ] with the aim of 2% enrichment. The priming dose of [1- 13 C]lysine was aiming for 1.5 mmol (1 h of infusion), and 1.28 mmol was the actual amount according to weight balance.

The solution of [ 13 C]bicarbonate was freshly prepared to minimize loss of 13 CO 2 . Specifically, [ 13 C]bicarbonate was weighed and dissolved in 20-mL 3-[methyl- 2 H 3 ]histidine solution in the morning of infusion day (Fig. 2 ). The 3-[methyl- 2 H 3 ]histidine (3MH) was used to estimate muscle protein breakdown, and the [ 13 C]bicarbonate was used to prime the CO 2 pool to accelerate the estimation of lysine oxidation rate. The primed-constant infusion of [1- 13 C]lysine was used to estimate lysine utilization by the mammary gland and the lysine flux in the whole body.

Timeline of isotope infusion and sampling (infusion day was within d 18–21)

Infusion protocol

The timeline for infusion is presented in Fig. 2 . Actual infusion day varied between d 14 and 18 due to real time patency of catheter. For lysine balance (Table 3 ) and body protein kinetics (Table 6 ), the actual infusion days were 17.0 ± 1.0 for CON and 17.0 ± 1.4 for OPT. For 3MH kinetics, the actual infusion days were 17.3 ± 1.0 for CON and 16.8 ± 1.5 for OPT. Pumps (Genie Touch TM , Kent Scientific Corp, Torrington, CT, USA) and syringes were placed on a large and stable plastic board laid above the farrowing stall. Following the priming dose, the infusion line was immediately attached to the syringe mounted to the pump to begin the constant infusion. The sampling line was coiled and stored in the pouch until used for blood sampling.

The mixed 20 mL saline solution containing 3-[methyl- 2 H 3 ]histidine (183 μmol) and [ 13 C]bicarbonate (368 μmol) was given through the infusion line as a bolus injection. The [ 13 C]bicarbonate in this infusate was used as a priming dose. After bolus injection, a constant 2-h infusion of [ 13 C]bicarbonate (368 μmol/h) began. The 2-h [ 13 C]bicarbonate infusion was followed by a 6-h primed constant [1- 13 C]lysine infusion (1.50 mmol/h) (Fig. 2 ).

Blood sampling

The timeline for blood sampling is presented in Fig. 2 . For analysis of plasma 3-[methyl- 2 H 3 ]histidine concentrations and estimation of muscle protein breakdown rate, blood samples were collected through the sampling line at 0 (immediately after termination of the bolus infusion), 5, 10, 15, 30 and 45 min and 1, 2, 3, 4, 5, 6, 7, 8, 24, 34, 48, 58 and 72 h post bolus infusion. Blood samples (0.5 mL) were transferred into 500-μL BD microtainer tubes (K 2 EDTA) and centrifuged (1,500 × g at 4 °C for 5 min). The plasma was extracted and stored in 1.5-mL microcentrifuge tubes at −20 °C until analysis.

For analysis of plasma [1- 13 C]lysine concentrations and estimation of whole-body Lys flux, blood samples (0.5 mL) were collected prior to infusion for background enrichment and at 1, 2, 3, 4, 5 and 6 h from the start of [1- 13 C]lysine infusion (Fig. 2 ).

For analysis of blood CO 2 concentrations, blood samples (2 mL) were collected prior to [ 13 C]bicarbonate-prime infusion for background, and at 1, 2, 3, 4, 5, 6, 7 and 8 h following the prime infusion. Blood samples were injected into evacuated vacutainer tubes (Becton Dickinson, Plymouth, UK) previously prepared with 2 mL of phosphoric acid, immediately mixed, and cooled to room temperature. The CO 2 was then transferred from evacuated vacutainers to Exetainer tubes (Labco Breath Tube, UK) by using pure nitrogen gas as medium until analysis.

Milk sampling protocol

The timeline for milk sampling is presented in Fig. 2 . Milk samples were taken between d 14 and 18 during the infusion protocol. Milk was sampled before infusion for background enrichment, and at 1, 2, 3, 4, 5 and 6 h of primed constant infusion of Lys.

For each milk sampling period, piglets were separated from the sows for 1 h in an empty adjacent farrowing crate with no access to water, and sows were administered 1 mL of oxytocin (20 IU/mL oxytocin, sodium chloride 0.9% w/v, and chlorobutanol 0.5% w/v, VetTek, Blue Springs, MO, USA) through the sampling catheter immediately after blood sampling. The catheter was rinsed with 2 mL of saline solution to ensure oxytocin reached the blood circulation. A total of 30-mL milk was manually collected across all glands and stored in 2 separate 15-mL tubes (polypropylene centrifuge tubes with screw cap, Denville Scientific, Swedesboro, NJ, USA). Piglets were immediately returned to sows to complete nursing and empty the mammary glands. Piglets were removed after nursing and kept separate from the sow until the next milk sampling time, 1 h later.

Isotope analysis

Plasma and milk [1- 13 C]lysine and 3-[methyl- 2 H 3 ]histidine (after acid hydrolysis) were determined as their dansyl derivatives by HESI LC-MS as previously described [ 18 ]. The following m/z transitions were monitored: 613→379 and 614→380 for [1- 13 C]lysine and 403→124 and 406→127 for 3-[methyl- 2 H 3 ]histidine. Determination of blood 13 CO 2 enrichment was performed by IRMS (Delta+XL IRMS coupled with GasBench-II peripheral device, Thermo-Quest Finnigan, Bremen, Germany) as previously described [ 19 ].

Nutrient analysis

Feed samples were analyzed for gross energy (GE) by bomb calorimetry (Parr Instrument Inc., Moline, IL, USA). Dry matter and N in feed samples were analyzed as previously described [ 5 ]. Dietary AA analysis [AOAC Official Method 982.30 E (a,b,c), 45.3.05, 2006] was performed by the Agricultural Experiment Station Chemical Laboratories (University of Missouri-Columbia, Columbia, MO, USA) as outlined in previous reports [ 5 ].

Calculations

The following assumptions were made during calculation:

Priming dose of isotope was assumed to mix with pool instantly.

The appearance of unlabeled bicarbonate was constant during the time of primed-constant infusion of bicarbonate (2 h) and that of [1- 13 C]lysine (6 h).

[1- 13 C]lysine cannot be synthesized once 1-carbon was lost to CO 2 , thus rate of lysine decarboxylation represented rate of lysine breakdown.

Kinetics of plasma lysine was an indicator of kinetics of whole body protein.

The indicator AA (lysine) was assumed to be oxidised for maintenance or incorporated into milk protein without other metabolic pathway.

Lysine oxidation

The enrichment of CO 2 during the period of primed-constant infusion of [ 13 C]bicarbonate was calculated as follows (Eq. 1 ):

Where “infusion \({\text{rate}}_{\text{H}{}^{13}\text{CO}_3^-}\) ” represents the infusion rate (368 μmol/h) of [ 13 C]bicarbonate, and “ \(\text{Ra}_{\text{HCO}_{3}^-}\) ” represents the rate of appearance of unlabeled bicarbonate (baseline) in the body.

The enrichment of CO 2 during the period of primed-constant infusion of [1- 13 C]lysine was calculated as follows (Eq. 2 ):

Where “ \({\text{Ra}}_{\text{H}{}^{13}\text{CO}_3^-}\) ” represents the rate of appearance of labeled bicarbonate from [1- 13 C]lysine oxidation, and “ \(\text{Ra}_{\text{HCO}_{3}^-}\) ” represents the rate of appearance of unlabeled bicarbonate (baseline) in the body as in Eq. 1 .

The enrichment of lysine during the period of primed-constant infusion of [1- 13 C]lysine was calculated as follows (Eq. 3 ):

Where Ra Lys represents the rate of appearance of unlabeled lysine in the body.

Lysine oxidation was estimated as follows (Eq. 4 ):

Whole body protein breakdown and synthesis

Whole body protein breakdown (PB) and synthesis (PS) were mirrored by Lys dynamics (Table 3 and Fig. 3 ).

Isotopic enrichment of [1- 13 C]lysine in plasma (panel a ) and milk (panel b ) over 6 h during peak lactation (d 14 to 18) for sows fed control (CON; 18.4% CP; n = 3) and optimal (OPT; 14.0% CP; n = 5) diets

The PB and PS were calculated as follows (Eqs. 5 and 6 ):

Where 146.19 g/mol is the molar weight of isotopic Lys, 6.74% is the average weight percentage of Lys in the sow’s body protein [ 6 ].

The average milk protein concentration of 5.16% was used [ 6 ]. Milk yield was estimated according to Theil et al. [ 15 ].

Muscle protein breakdown

Data was expressed as tracer to tracee ratio (TTR). Multiexponential models were fitted to the data (Eq. 10 and Fig. 1 ). Residual inspection and pseudo-R 2 were used to determine the most parsimonious model that best fitted the data from each individual sow. Area under the curve (AUC; TTR•h) was calculated using the parameters from the multiexponential equation (Eq. 11 and Table 4 ) and 3MH rate of appearance (Ra; μmol/kg/h) was calculated by dividing the dose administered (μmol/kg) by AUC (Eq. 12 and Table 4 ). Half-life (h) was determined using the rate constant corresponding to the tail of the curve (Eq. 13 , Table 4 , and Fig. 1 ).

Muscle protein breakdown rate (%/d) was calculated as follows (Eq. 14 ):

Where total protein bound 3MH pool = muscle protein mass (g) × 3.8742 μmol 3MH/g protein and muscle protein mass = 8.21% × sow BW (kg) [ 20 ].

Muscle protein breakdown (g/d), was calculated as follows (Eq. 15 ):

Efficiency of lysine for lactation

Lysine utilization efficiency for lactation was calculated as follows (Eq. 16 ):

Statistical analysis

Data were confirmed for homogeneity of residual variance and normality of residuals by Mixed Procedures and Univariate Procedures of SAS 9.4 (SAS Inst. Inc., Cary, NC, USA) before ANOVA analysis (Mixed model procedures).

For the lysine balance and protein kinetic estimation, two sows from CON were removed from the data set. In one case, both ear vein catheters lost patency at the time of infusion, and in the other case, one of the 2 ear vein catheters lost patency. The latter sow, however, was used for the estimation of muscle protein breakdown, which only required one catheter. Therefore, the number of sows for the lysine balance data and protein kinetic estimation were 5 and 3 for OPT and CON, respectively.

For the analysis of lysine enrichment in plasma and milk, the following model was used:

The Enrichment of lysine depended on the fixed effects of diet ( OPT vs. CON ) , and sampling hour, with hour included as repeated measurement. The random effects included block and individual sow . The interactive effect of diet × hour was also included .

For the analysis of lysine balance, body protein breakdown and synthesis, and muscle protein breakdown rate, identified as “Response”, the following model was used:

The Response depended on the fixed effects of diet ( OPT vs. CON ) . The random effects included block and individual sow .

Differences between treatments were declared at P < 0.05 and tendencies at P ≤ 0.1.

Lactation performance

Lactation performance during the 21-d period and milk yield and nutrient concentrations between d 14 and 18 are presented in Table 5 . Sow initial BW and ADFI did not differ between OPT and CON diets. Litter growth rate of sows fed OPT diet was greater than those fed CON diet ( P < 0.05).

Lysine balance and efficiency of utilization

Lysine balance values are presented in Table 3 . The SID Lys intake, Lys oxidation, flux and Lys associated with protein synthesis did not differ between OPT and CON diets (Table 3 ). Compared to sows fed CON, those fed OPT had greater efficiency of Lys (0.62 vs. 0.50; P < 0.05) and tended to have a lower ( P = 0.069) released Lys associated with protein breakdown.

Whole body protein synthesis, whole body protein breakdown and fractional muscle protein breakdown

Whole body protein breakdown rate and synthesis rate tended to be lower ( P = 0.069 and P = 0.109, respectively) and protein turnover efficiency (synthesis: breakdown) tended to be greater ( P = 0.060) in sows fed OPT compared to those fed CON (Table 6 ). Whole body protein net synthesis (i.e., whole body protein synthesis − whole body protein breakdown) did not differ between OPT and CON diets.

For estimation of muscle protein breakdown rate, an additional sow in OPT treatment lost patency of both catheters, therefore the number of sows was 4 in each of the treatment. A 3-exponential model best fitted the 3MH decaying curve (Fig. 1 ) and pseudo- R 2 were > 0.995. Muscle protein breakdown rate and fractional muscle protein breakdown rate (%) did not differ ( P = 0.197) between sows fed OPT and CON diets (4.84% and 5.59%, respectively) (Table 6 ).

Enrichment of lysine

Lysine enrichment in plasma (panel a) and milk (panel b) is presented in Fig. 3 . The lysine enrichment in plasma did not differ between diets and time. Lysine enrichment in milk was lower ( P < 0.01) in sows fed OPT compared to sows fed CON diets and differed over time ( P < 0.01). There was no interaction between diets and time.

fDynamics of 3-[methyl- 2 H 3 ]histidine

Plasma isotopic enrichment of 3-MH following 3-MH bolus infusion is presented in Fig. 1 , and relevant dynamic parameters are presented in Table 4 . Plasma isotopic enrichment of 3-MH of sows fed CON was lower ( P < 0.001) than that for OPT diet. Time effects of 3-MH were significant ( P < 0.001) in both treatments of CON and OPT, and no interaction effect between diet and time ( P = 0.894) was detected.

Previous studies showed that lactating sows fed low CP diets with CAA supplementation had greater milk casein yield [ 2 , 3 ], and utilization efficiency of N and EAA [ 4 , 5 ]. The improvement of milk yield however was at the expense of sow BW and maternal N retention [ 3 , 5 ]. Zhang et al. [ 8 ] suggested that feeding diets with improved AA balance triggered nutrient repartitioning to milk at the expense of maternal adipose tissue rather than protein tissue. Maternal body fat loss affects subsequent reproductive performance and compromises the overall production efficiency during the sow’s life span [ 21 ]. Therefore, commercial implementation of diets with aggressing reduction in CP with CAA supplementation to achieve improved AA balance will not only depend on their impact on lactation performance and production efficiency but also on ensuring that long-term maternal body protein and lipid reserves are not compromised.

The mechanisms behind the reduced maternal N retention in sows fed improved AA balance diets reported in earlier studies [ 4 , 5 ] are unclear. Reduced maternal body protein synthesis, greater body protein breakdown, or a combination of thereof can dictate maternal N balance during lactation. In this study however, BW and backfat change during lactation did not differ between OPT and CON sows. Of note, sows fed OPT had no change in BW with a small loss in backfat while sows fed CON gained 5.5 kg with no change in backfat. Body protein kinetics in this study (Table 6 ) dictated that whole body protein net synthesis (whole body protein synthesis − whole body protein breakdown) of sows fed CON and OPT were close (1,041.72 vs. 1,022.90 g/d), but note that whole body protein net synthesis of lactating sows included milk protein yield and maternal protein deposition. Milk protein yield was greater in OPT than CON as mirrored by litter growth rate (Table 5 ). Consequently, maternal protein deposition was greater in CON than OPT which aligns with the observation that body weight increased in sows fed CON while there was no change of body weight in sows fed OPT (Table 5 ). In addition, increased milk production in sows fed OPT suggest that OPT diet led sows to partition more dietary nutrient towards milk than maternal reserves, in other words, sows fed OPT were more motivated to produce milk even at the expense of maternal deposition.

This study used Lys as representative AA of body protein to analyze whole body protein turnover. In essence, Lys flux in the blood was contributed by dietary Lys intake and Lys released by body protein breakdown, and free Lys in the blood could be directed to either Lys oxidation or Lys incorporation into body protein (Fig. 4 ). Thus, body protein breakdown and synthesis could be estimated by measuring Lys flux in blood and Lys oxidation. The carbon dioxide released by Lys oxidation remains in the blood bicarbonate pool and mixed with carbon dioxide from other substrate oxidation (Fig. 5 ). By priming the bicarbonate pool, the baseline production rate of carbon dioxide can be estimated based on bicarbonate enrichment and constant infusion rate of labeled bicarbonate during prime-constant infusion of bicarbonate (Eq. 1 ). The release of labeled carbon dioxide due to labeled Lys oxidation was proportional to the baseline production rate of carbon dioxide according to enrichment of bicarbonate during prime-constant infusion of Lys (Eqs. 2 and  3 ).

Diagram of lysine balance in lactating sows at fed state

Representation of a two-pool model to estimate lysine oxidation

Milk protein synthesis represents the difference between whole body protein synthesis and breakdown, assuming that maternal protein retention is close to zero, since maternal tissue mobilization is majorly comprised of body fat rather than body protein [ 5 , 8 ]. According to this assumption, milk protein output rate measured by isotopic technique (Eq. 8 ) was 1,023 to 1,042 g/d, which aligns well with a previous study where 957 g/d milk protein synthesis was reported using a N balance approach [ 5 ]. When compared to traditional method where milk protein synthesis is the product of milk yield and milk protein concentration (645 to 675 g/d; Eq. 9 ), the isotopic-predicted milk protein synthesis (1,023 to 1,042 g/d) appears overestimated. Guan et al. [ 7 ] reported milk protein synthesis of 575 g/d as the net balance between sow whole mammary protein synthesis (975 g/d) and breakdown rate (400 g/d), corroborating the values reported here (645–675 g/d) using the traditional method. Nitrogen balance techniques tend to overestimate actual nitrogen retention [ 22 , 23 ], as observed herein with the isotope technique (Eq. 8 ). Note that the estimated muscle protein breakdown rate according to 3MH method in this study was 960 to 1,261 g/d (Eq. 15 ), which was greater than the protein breakdown rate (650 to 1,051 g/d; Eq. 8 ) based on the Lys flux. On the other hand, milk protein synthesis rate per metabolic BW (BW 0.75 ) were 10.25 and 9.85 g/d/kg 0.75 for OPT and CON, respectively in this study, supporting a previously reported value of 11.57 g/d/kg 0.75 (using Val as representative AA) [ 24 ]. Thus, overestimation of milk protein synthesis (Eq. 8 ) was majorly attributed to an underestimation of protein breakdown rather than overestimation of protein synthesis. The underestimation of body protein breakdown according to Lys flux (Eq. 5 ) may be partially due to the tendency to overestimate feed intake [ 23 ], although feed waste was minimized in this study. Nevertheless, it is also important to note that estimated muscle protein breakdown (15.4 to 17.8 μmol/kg/d; Eq. 15 ; Table 6 ) and fractional breakdown (4.84–5.59%/d; Eq. 14 ; Table 6 ) in this study was greater than those reported for lactating gilt (3.4%/d, 12.0 μmol/kg/d) using the same 3MH method [ 17 ]. It is speculated that the multiparous lactating sow may mobilize body protein more readily compared to the lactating gilt.

In this study, milk protein yield of lactating sows fed OPT diet did not differ from those fed control diet neither when an isotopic method nor the traditional method were used. Although there was no difference between whole body protein synthesis and breakdown, the absolute values of protein synthesis and breakdown were both lower in sows fed OPT diet compared to CON diet, suggesting less whole-body protein turnover in sows fed the OPT diet. In support of this view, previous studies also showed a decreased protein breakdown rate reflected by lower urea nitrogen output when sows were fed reduced protein diets [ 3 , 5 ]. The biological process of protein turnover is energetically costly [ 1 , 25 ]. Zhang et al. [ 5 ] reported feeding sows with an improved AA balance diet was associated with higher energy efficiency, lending support to the current observation.

The Lys efficiency for sows fed different levels of dietary protein based on the NRC [ 6 ] approach was previously determined [ 4 , 5 ], with greater efficiency values (0.68 and 0.66, respectively) found during peak lactation (d 14–18) in sows fed low CP diets balanced with CAA. Herein, greater Lys utilization efficiency values, determined using a different approach, were also found in sows fed OPT (0.62) compared to CON (0.50). The estimation of Lys utilization efficiency was based on Lys balance parameters, i.e., Lys flux in blood, SID Lys intake and Lys oxidation (Fig. 4 ), and the assumption that net protein synthesis (protein synthesis − protein breakdown) represents milk protein synthesis, with negligible maternal body retention. The true Lys utilization efficiency is the ratio between “Lys in milk” and “Lys for milk”, thus Lys utilized for maintenance should be excluded in the denominator [ 6 ] as follows:

In this study, the whole-body Lys flux was corrected by excluding Lys oxidation (Eq. 16 ), which corresponds to the Lys requirement for maintenance. Guan et al. [ 7 ] reported that Lys flux partitioned to the mammary glands as percentage of whole-body Lys flux was 56% in sows fed a conventional diet, which is comparable to the Lys efficiency values of 50% to 62% observed in this study.

Feeding lactation sows with an improved AA balance diet did not affect milk protein yield and reduced whole-body protein turnover. The reduced whole-body protein turnover resulted from a decrease in both whole-body protein synthesis and breakdown rate, with a tendency for greater protein synthesis to protein breakdown ratio (2.65 vs. 2.02).

Efficiency of Lys was also greater during peak lactation, together suggesting higher efficiency of energy use. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

Availability of data and materials

All data generated or analyzed during this study are available from the corresponding author on request.

Abbreviations

Average daily gain

Area under the curve

Body weight

Crystalline amino acid

Crude protein

Essential amino acid

Standard error of the mean

Standardized ileal digestibility

3-[methyl- 2 H 3 ]histidine

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This work was financially supported by funds from the USDA-NIFA (award number 2014-67015-21832).

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Sai Zhang, Julie Moore & Nathalie L. Trottier

State Key Laboratory of Swine and Poultry Breeding Industry, Key Laboratory of Animal Nutrition and Feed Science in South China, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Animal Breeding and Nutrition, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, PR China

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Juan C. Marini & Mahmoud A. Mohammad

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Zhang, S., Marini, J.C., Mavangira, V. et al. Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows. J Animal Sci Biotechnol 15 , 65 (2024). https://doi.org/10.1186/s40104-024-01020-9

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The Flaws and Human Harms of Animal Experimentation

Nonhuman animal (“animal”) experimentation is typically defended by arguments that it is reliable, that animals provide sufficiently good models of human biology and diseases to yield relevant information, and that, consequently, its use provides major human health benefits. I demonstrate that a growing body of scientific literature critically assessing the validity of animal experimentation generally (and animal modeling specifically) raises important concerns about its reliability and predictive value for human outcomes and for understanding human physiology. The unreliability of animal experimentation across a wide range of areas undermines scientific arguments in favor of the practice. Additionally, I show how animal experimentation often significantly harms humans through misleading safety studies, potential abandonment of effective therapeutics, and direction of resources away from more effective testing methods. The resulting evidence suggests that the collective harms and costs to humans from animal experimentation outweigh potential benefits and that resources would be better invested in developing human-based testing methods.

Introduction

Annually, more than 115 million animals are used worldwide in experimentation or to supply the biomedical industry. 1 Nonhuman animal (hereafter “animal”) experimentation falls under two categories: basic (i.e., investigation of basic biology and human disease) and applied (i.e., drug research and development and toxicity and safety testing). Regardless of its categorization, animal experimentation is intended to inform human biology and health sciences and to promote the safety and efficacy of potential treatments. Despite its use of immense resources, the animal suffering involved, and its impact on human health, the question of animal experimentation’s efficacy has been subjected to little systematic scrutiny. 2

Although it is widely accepted that medicine should be evidence based , animal experimentation as a means of informing human health has generally not been held, in practice, to this standard. This fact makes it surprising that animal experimentation is typically viewed as the default and gold standard of preclinical testing and is generally supported without critical examination of its validity. A survey published in 2008 of anecdotal cases and statements given in support of animal experimentation demonstrates how it has not and could not be validated as a necessary step in biomedical research, and the survey casts doubt on its predictive value. 3 I show that animal experimentation is poorly predictive of human outcomes, 4 that it is unreliable across a wide category of disease areas, 5 and that existing literature demonstrates the unreliability of animal experimentation, thereby undermining scientific arguments in its favor. I further show that the collective harms that result from an unreliable practice tip the ethical scale of harms and benefits against continuation in much, if not all, of experimentation involving animals. 6

Problems of Successful Translation to Humans of Data from Animal Experimentation

Although the unreliability and limitations of animal experimentation have increasingly been acknowledged, there remains a general confidence within much of the biomedical community that they can be overcome. 7 However, three major conditions undermine this confidence and explain why animal experimentation, regardless of the disease category studied, fails to reliably inform human health: (1) the effects of the laboratory environment and other variables on study outcomes, (2) disparities between animal models of disease and human diseases, and (3) species differences in physiology and genetics. I argue for the critical importance of each of these conditions.

The Influence of Laboratory Procedures and Environments on Experimental Results

Laboratory procedures and conditions exert influences on animals’ physiology and behaviors that are difficult to control and that can ultimately impact research outcomes. Animals in laboratories are involuntarily placed in artificial environments, usually in windowless rooms, for the duration of their lives. Captivity and the common features of biomedical laboratories—such as artificial lighting, human-produced noises, and restricted housing environments—can prevent species-typical behaviors, causing distress and abnormal behaviors among animals. 8 Among the types of laboratory-generated distress is the phenomenon of contagious anxiety. 9 Cortisone levels rise in monkeys watching other monkeys being restrained for blood collection. 10 Blood pressure and heart rates elevate in rats watching other rats being decapitated. 11 Routine laboratory procedures, such as catching an animal and removing him or her from the cage, in addition to the experimental procedures, cause significant and prolonged elevations in animals’ stress markers. 12 These stress-related changes in physiological parameters caused by the laboratory procedures and environments can have significant effects on test results. 13 Stressed rats, for example, develop chronic inflammatory conditions and intestinal leakage, which add variables that can confound data. 14

A variety of conditions in the laboratory cause changes in neurochemistry, genetic expression, and nerve regeneration. 15 In one study, for example, mice were genetically altered to develop aortic defects. Yet, when the mice were housed in larger cages, those defects almost completely disappeared. 16 Providing further examples, typical noise levels in laboratories can damage blood vessels in animals, and even the type of flooring on which animals are tested in spinal cord injury experiments can affect whether a drug shows a benefit. 17

In order to control for potential confounders, some investigators have called for standardization of laboratory settings and procedures. 18 One notable effort was made by Crabbe et al. in their investigation of the potential confounding influences of the laboratory environment on six mouse behaviors that are commonly studied in neurobehavioral experiments. Despite their “extraordinary lengths to equate test apparatus, testing protocols, and all possible features of animal husbandry” across three laboratories, there were systematic differences in test results in these labs. 19 Additionally, different mouse strains varied markedly in all behavioral tests, and for some tests the magnitude of genetic differences depended on the specific testing laboratory. The results suggest that there are important influences of environmental conditions and procedures specific to individual laboratories that can be difficult—perhaps even impossible—to eliminate. These influences can confound research results and impede extrapolation to humans.

The Discordance between Human Diseases and Animal Models of Diseases

The lack of sufficient congruence between animal models and human diseases is another significant obstacle to translational reliability. Human diseases are typically artificially induced in animals, but the enormous difficulty of reproducing anything approaching the complexity of human diseases in animal models limits their usefulness. 20 Even if the design and conduct of an animal experiment are sound and standardized, the translation of its results to the clinic may fail because of disparities between the animal experimental model and the human condition. 21

Stroke research presents one salient example of the difficulties in modeling human diseases in animals. Stroke is relatively well understood in its underlying pathology. Yet accurately modeling the disease in animals has proven to be an exercise in futility. To address the inability to replicate human stroke in animals, many assert the need to use more standardized animal study design protocols. This includes the use of animals who represent both genders and wide age ranges, who have comorbidities and preexisting conditions that occur naturally in humans, and who are consequently given medications that are indicated for human patients. 22 In fact, a set of guidelines, named STAIR, was implemented by a stroke roundtable in 1999 (and updated in 2009) to standardize protocols, limit the discrepancies, and improve the applicability of animal stroke experiments to humans. 23 One of the most promising stroke treatments later to emerge was NXY-059, which proved effective in animal experiments. However, the drug failed in clinical trials, despite the fact that the set of animal experiments on this drug was considered the poster child for the new experimental standards. 24 Despite such vigorous efforts, the development of STAIR and other criteria has yet to make a recognizable impact in clinical translation. 25

Under closer scrutiny, it is not difficult to surmise why animal stroke experiments fail to successfully translate to humans even with new guidelines. Standard stroke medications will likely affect different species differently. There is little evidence to suggest that a female rat, dog, or monkey sufficiently reproduces the physiology of a human female. Perhaps most importantly, reproducing the preexisting conditions of stroke in animals proves just as difficult as reproducing stroke pathology and outcomes. For example, most animals don’t naturally develop significant atherosclerosis, a leading contributor to ischemic stroke. In order to reproduce the effects of atherosclerosis in animals, researchers clamp their blood vessels or artificially insert blood clots. These interventions, however, do not replicate the elaborate pathology of atherosclerosis and its underlying causes. Reproducing human diseases in animals requires reproducing the predisposing diseases, also a formidable challenge. The inability to reproduce the disease in animals so that it is congruent in relevant respects with human stroke has contributed to a high failure rate in drug development. More than 114 potential therapies initially tested in animals failed in human trials. 26

Further examples of repeated failures based on animal models include drug development in cancer, amyotrophic lateral sclerosis (ALS), traumatic brain injury (TBI), Alzheimer’s disease (AD), and inflammatory conditions. Animal cancer models in which tumors are artificially induced have been the basic translational model used to study key physiological and biochemical properties in cancer onset and propagation and to evaluate novel treatments. Nevertheless, significant limitations exist in the models’ ability to faithfully mirror the complex process of human carcinogenesis. 27 These limitations are evidenced by the high (among the highest of any disease category) clinical failure rate of cancer drugs. 28 Analyses of common mice ALS models demonstrate significant differences from human ALS. 29 The inability of animal ALS models to predict beneficial effects in humans with ALS is recognized. 30 More than twenty drugs have failed in clinical trials, and the only U.S. Food and Drug Administration (FDA)–approved drug to treat ALS is Riluzole, which shows notably marginal benefit on patient survival. 31 Animal models have also been unable to reproduce the complexities of human TBI. 32 In 2010, Maas et al. reported on 27 large Phase 3 clinical trials and 6 unpublished trials in TBI that all failed to show human benefit after showing benefit in animals. 33 Additionally, even after success in animals, around 172 and 150 drug development failures have been identified in the treatment of human AD 34 and inflammatory diseases, 35 respectively.

The high clinical failure rate in drug development across all disease categories is based, at least in part, on the inability to adequately model human diseases in animals and the poor predictability of animal models. 36 A notable systematic review, published in 2007, compared animal experimentation results with clinical trial findings across interventions aimed at the treatment of head injury, respiratory distress syndrome, osteoporosis, stroke, and hemorrhage. 37 The study found that the human and animal results were in accordance only half of the time. In other words, the animal experiments were no more likely than a flip of the coin to predict whether those interventions would benefit humans.

In 2004, the FDA estimated that 92 percent of drugs that pass preclinical tests, including “pivotal” animal tests, fail to proceed to the market. 38 More recent analysis suggests that, despite efforts to improve the predictability of animal testing, the failure rate has actually increased and is now closer to 96 percent. 39 The main causes of failure are lack of effectiveness and safety problems that were not predicted by animal tests. 40

Usually, when an animal model is found wanting, various reasons are proffered to explain what went wrong—poor methodology, publication bias, lack of preexisting disease and medications, wrong gender or age, and so on. These factors certainly require consideration, and recognition of each potential difference between the animal model and the human disease motivates renewed efforts to eliminate these differences. As a result, scientific progress is sometimes made by such efforts. However, the high failure rate in drug testing and development, despite attempts to improve animal testing, suggests that these efforts remain insufficient to overcome the obstacles to successful translation that are inherent to the use of animals. Too often ignored is the well-substantiated idea that these models are, for reasons summarized here, intrinsically lacking in relevance to, and thus highly unlikely to yield useful information about, human diseases. 41

Interspecies Differences in Physiology and Genetics

Ultimately, even if considerable congruence were shown between an animal model and its corresponding human disease, interspecies differences in physiology, behavior, pharmacokinetics, and genetics would significantly limit the reliability of animal studies, even after a substantial investment to improve such studies. In spinal cord injury, for example, drug testing results vary according to which species and even which strain within a species is used, because of numerous interspecies and interstrain differences in neurophysiology, anatomy, and behavior. 42 The micropathology of spinal cord injury, injury repair mechanisms, and recovery from injury varies greatly among different strains of rats and mice. A systematic review found that even among the most standardized and methodologically superior animal experiments, testing results assessing the effectiveness of methylprednisolone for spinal cord injury treatment varied considerably among species. 43 This suggests that factors inherent to the use of animals account for some of the major differences in results.

Even rats from the same strain but purchased from different suppliers produce different test results. 44 In one study, responses to 12 different behavioral measures of pain sensitivity, which are important markers of spinal cord injury, varied among 11 strains of mice, with no clear-cut patterns that allowed prediction of how each strain would respond. 45 These differences influenced how the animals responded to the injury and to experimental therapies. A drug might be shown to help one strain of mice recover but not another. Despite decades of using animal models, not a single neuroprotective agent that ameliorated spinal cord injury in animal tests has proven efficacious in clinical trials to date. 46

Further exemplifying the importance of physiological differences among species, a 2013 study reported that the mouse models used extensively to study human inflammatory diseases (in sepsis, burns, infection, and trauma) have been misleading. The study found that mice differ greatly from humans in their responses to inflammatory conditions. Mice differed from humans in what genes were turned on and off and in the timing and duration of gene expression. The mouse models even differed from one another in their responses. The investigators concluded that “our study supports higher priority to focus on the more complex human conditions rather than relying on mouse models to study human inflammatory disease.” 47 The different genetic responses between mice and humans are likely responsible, at least in part, for the high drug failure rate. The authors stated that every one of almost 150 clinical trials that tested candidate agents’ ability to block inflammatory responses in critically ill patients failed.

Wide differences have also become apparent in the regulation of the same genes, a point that is readily seen when observing differences between human and mouse livers. 48 Consistent phenotypes (observable physical or biochemical characteristics) are rarely obtained by modification of the same gene, even among different strains of mice. 49 Gene regulation can substantially differ among species and may be as important as the presence or absence of a specific gene. Despite the high degree of genome conservation, there are critical differences in the order and function of genes among species. To use an analogy: as pianos have the same keys, humans and other animals share (largely) the same genes. Where we mostly differ is in the way the genes or keys are expressed. For example, if we play the keys in a certain order, we hear Chopin; in a different order, we hear Ray Charles; and in yet a different order, it’s Jerry Lee Lewis. In other words, the same keys or genes are expressed, but their different orders result in markedly different outcomes.

Recognizing the inherent genetic differences among species as a barrier to translation, researches have expressed considerable enthusiasm for genetically modified (GM) animals, including transgenic mice models, wherein human genes are inserted into the mouse genome. However, if a human gene is expressed in mice, it will likely function differently from the way it functions in humans, being affected by physiological mechanisms that are unique in mice. For example, a crucial protein that controls blood sugar in humans is missing in mice. 50 When the human gene that makes this protein was expressed in genetically altered mice, it had the opposite effect from that in humans: it caused loss of blood sugar control in mice. Use of GM mice has failed to successfully model human diseases and to translate into clinical benefit across many disease categories. 51 Perhaps the primary reason why GM animals are unlikely to be much more successful than other animal models in translational medicine is the fact that the “humanized” or altered genes are still in nonhuman animals.

In many instances, nonhuman primates (NHPs) are used instead of mice or other animals, with the expectation that NHPs will better mimic human results. However, there have been sufficient failures in translation to undermine this optimism. For example, NHP models have failed to reproduce key features of Parkinson’s disease, both in function and in pathology. 52 Several therapies that appeared promising in both NHPs and rat models of Parkinson’s disease showed disappointing results in humans. 53 The campaign to prescribe hormone replacement therapy (HRT) in millions of women to prevent cardiovascular disease was based in large part on experiments on NHPs. HRT is now known to increase the risk of these diseases in women. 54

HIV/AIDS vaccine research using NHPs represents one of the most notable failures in animal experimentation translation. Immense resources and decades of time have been devoted to creating NHP (including chimpanzee) models of HIV. Yet all of about 90 HIV vaccines that succeeded in animals failed in humans. 55 After HIV vaccine gp120 failed in clinical trials, despite positive outcomes in chimpanzees, a BMJ article commented that important differences between NHPs and humans with HIV misled researchers, taking them down unproductive experimental paths. 56 Gp120 failed to neutralize HIV grown and tested in cell culture. However, because the serum protected chimpanzees from HIV infection, two Phase 3 clinical trials were undertaken 57 —a clear example of how expectations that NHP data are more predictive than data from other (in this case, cell culture) testing methods are unproductive and harmful. Despite the repeated failures, NHPs (though not chimpanzees or other great apes) remain widely used for HIV research.

The implicit assumption that NHP (and indeed any animal) data are reliable has also led to significant and unjustifiable human suffering. For example, clinical trial volunteers for gp120 were placed at unnecessary risk of harm because of unfounded confidence in NHP experiments. Two landmark studies involving thousands of menopausal women being treated with HRT were terminated early because of increased stroke and breast cancer risk. 58 In 2003, Elan Pharmaceuticals was forced to prematurely terminate a Phase 2 clinical trial when an investigational AD vaccine was found to cause brain swelling in human subjects. No significant adverse effects were detected in GM mice or NHPs. 59

In another example of human suffering resulting from animal experimentation, six human volunteers were injected with an immunomodulatory drug, TGN 1412, in 2006. 60 Within minutes of receiving the experimental drug, all volunteers suffered a severe adverse reaction resulting from a life-threatening cytokine storm that led to catastrophic systemic organ failure. The compound was designed to dampen the immune system, but it had the opposite effect in humans. Prior to this first human trial, TGN 1412 was tested in mice, rabbits, rats, and NHPs with no ill effects. NHPs also underwent repeat-dose toxicity studies and were given 500 times the human dose for at least four consecutive weeks. 61 None of the NHPs manifested the ill effects that humans showed almost immediately after receiving minute amounts of the test drug. Cynomolgus and rhesus monkeys were specifically chosen because their CD28 receptors demonstrated similar affinity to TGN 1412 as human CD28 receptors. Based on such data as these, it was confidently concluded that results obtained from these NHPs would most reliably predict drug responses in humans—a conclusion that proved devastatingly wrong.

As exemplified by the study of HIV/AIDS, TGN 1412, and other experiences, 62 experiments with NHPs are not necessarily any more predictive of human responses than experiments with other animals. The repeated failures in translation from studies with NHPs belie arguments favoring use of any nonhuman species to study human physiology and diseases and to test potential treatments. If experimentation using chimpanzees and other NHPs, our closest genetic cousins, are unreliable, how can we expect research using other animals to be reliable? The bottom line is that animal experiments, no matter the species used or the type of disease research undertaken, are highly unreliable—and they have too little predictive value to justify the resultant risks of harms for humans, for reasons I now explain.

The Collective Harms That Result from Misleading Animal Experiments

As medical research has explored the complexities and subtle nuances of biological systems, problems have arisen because the differences among species along these subtler biological dimensions far outweigh the similarities , as a growing body of evidence attests. These profoundly important—and often undetected—differences are likely one of the main reasons human clinical trials fail. 63

“Appreciation of differences” and “caution” about extrapolating results from animals to humans are now almost universally recommended. But, in practice, how does one take into account differences in drug metabolism, genetics, expression of diseases, anatomy, influences of laboratory environments, and species- and strain-specific physiologic mechanisms—and, in view of these differences, discern what is applicable to humans and what is not? If we cannot determine which physiological mechanisms in which species and strains of species are applicable to humans (even setting aside the complicating factors of different caging systems and types of flooring), the usefulness of the experiments must be questioned.

It has been argued that some information obtained from animal experiments is better than no information. 64 This thesis neglects how misleading information can be worse than no information from animal tests. The use of nonpredictive animal experiments can cause human suffering in at least two ways: (1) by producing misleading safety and efficacy data and (2) by causing potential abandonment of useful medical treatments and misdirecting resources away from more effective testing methods.

Humans are harmed because of misleading animal testing results. Imprecise results from animal experiments may result in clinical trials of biologically faulty or even harmful substances, thereby exposing patients to unnecessary risk and wasting scarce research resources. 65 Animal toxicity studies are poor predictors of toxic effects of drugs in humans. 66 As seen in some of the preceding examples (in particular, stroke, HRT, and TGN1412), humans have been significantly harmed because investigators were misled by the safety and efficacy profile of a new drug based on animal experiments. 67 Clinical trial volunteers are thus provided with raised hopes and a false sense of security because of a misguided confidence in efficacy and safety testing using animals.

An equal if indirect source of human suffering is the opportunity cost of abandoning promising drugs because of misleading animal tests. 68 As candidate drugs generally proceed down the development pipeline and to human testing based largely on successful results in animals 69 (i.e., positive efficacy and negative adverse effects), drugs are sometimes not further developed due to unsuccessful results in animals (i.e., negative efficacy and/or positive adverse effects). Because much pharmaceutical company preclinical data are proprietary and thus publicly unavailable, it is difficult to know the number of missed opportunities due to misleading animal experiments. However, of every 5,000–10,000 potential drugs investigated, only about 5 proceed to Phase 1 clinical trials. 70 Potential therapeutics may be abandoned because of results in animal tests that do not apply to humans. 71 Treatments that fail to work or show some adverse effect in animals because of species-specific influences may be abandoned in preclinical testing even if they may have proved effective and safe in humans if allowed to continue through the drug development pipeline.

An editorial in Nature Reviews Drug Discovery describes cases involving two drugs in which animal test results from species-specific influences could have derailed their development. In particular, it describes how tamoxifen, one of the most effective drugs for certain types of breast cancer, “would most certainly have been withdrawn from the pipeline” if its propensity to cause liver tumor in rats had been discovered in preclinical testing rather than after the drug had been on the market for years. 72 Gleevec provides another example of effective drugs that could have been abandoned based on misleading animal tests: this drug, which is used to treat chronic myelogenous leukemia (CML), showed serious adverse effects in at least five species tested, including severe liver damage in dogs. However, liver toxicity was not detected in human cell assays, and clinical trials proceeded, which confirmed the absence of significant liver toxicity in humans. 73 Fortunately for CML patients, Gleevec is a success story of predictive human-based testing. Many useful drugs that have safely been used by humans for decades, such as aspirin and penicillin, may not have been available today if the current animal testing regulatory requirements were in practice during their development. 74

A further example of near-missed opportunities is provided by experiments on animals that delayed the acceptance of cyclosporine, a drug widely and successfully used to treat autoimmune disorders and prevent organ transplant rejection. 75 Its immunosuppressive effects differed so markedly among species that researchers judged that the animal results limited any direct inferences that could be made to humans. Providing further examples, PharmaInformatic released a report describing how several blockbuster drugs, including aripiprazole (Abilify) and esomeprazole (Nexium), showed low oral bioavailability in animals. They would likely not be available on the market today if animal tests were solely relied on. Understanding the implications of its findings for drug development in general, PharmaInformatic asked, “Which other blockbuster drugs would be on the market today, if animal trials would have not been used to preselect compounds and drug-candidates for further development?” 76 These near-missed opportunities and the overall 96 percent failure rate in clinical drug testing strongly suggest the unsoundness of animal testing as a precondition of human clinical trials and provide powerful evidence for the need for a new, human-based paradigm in medical research and drug development.

In addition to potentially causing abandonment of useful treatments, use of an invalid animal disease model can lead researchers and the industry in the wrong research direction, wasting time and significant investment. 77 Repeatedly, researchers have been lured down the wrong line of investigation because of information gleaned from animal experiments that later proved to be inaccurate, irrelevant, or discordant with human biology. Some claim that we do not know which benefits animal experiments, particularly in basic research, may provide down the road. Yet human lives remain in the balance, waiting for effective therapies. Funding must be strategically invested in the research areas that offer the most promise.

The opportunity costs of continuing to fund unreliable animal tests may impede development of more accurate testing methods. Human organs grown in the lab, human organs on a chip, cognitive computing technologies, 3D printing of human living tissues, and the Human Toxome Project are examples of new human-based technologies that are garnering widespread enthusiasm. The benefit of using these testing methods in the preclinical setting over animal experiments is that they are based on human biology. Thus their use eliminates much of the guesswork required when attempting to extrapolate physiological data from other species to humans. Additionally, these tests offer whole-systems biology, in contrast to traditional in vitro techniques. Although they are gaining momentum, these human-based tests are still in their relative infancy, and funding must be prioritized for their further development. The recent advancements made in the development of more predictive, human-based systems and biological approaches in chemical toxicological testing are an example of how newer and improved tests have been developed because of a shift in prioritization. 78 Apart from toxicology, though, financial investment in the development of human-based technologies generally falls far short of investment in animal experimentation. 79

The unreliability of applying animal experimental results to human biology and diseases is increasingly recognized. Animals are in many respects biologically and psychologically similar to humans, perhaps most notably in the shared characteristics of pain, fear, and suffering. 80 In contrast, evidence demonstrates that critically important physiological and genetic differences between humans and other animals can invalidate the use of animals to study human diseases, treatments, pharmaceuticals, and the like. In significant measure, animal models specifically, and animal experimentation generally, are inadequate bases for predicting clinical outcomes in human beings in the great bulk of biomedical science. As a result, humans can be subject to significant and avoidable harm.

The data showing the unreliability of animal experimentation and the resultant harms to humans (and nonhumans) undermine long-standing claims that animal experimentation is necessary to enhance human health and therefore ethically justified. Rather, they demonstrate that animal experimentation poses significant costs and harms to human beings. It is possible—as I have argued elsewhere—that animal research is more costly and harmful, on the whole, than it is beneficial to human health. 81 When considering the ethical justifiability of animal experiments, we should ask if it is ethically acceptable to deprive humans of resources, opportunity, hope, and even their lives by seeking answers in what may be the wrong place. In my view, it would be better to direct resources away from animal experimentation and into developing more accurate, human-based technologies.

Aysha Akhtar , M.D., M.P.H., is a neurologist and preventive medicine specialist and Fellow at the Oxford Centre for Animal Ethics, Oxford, United Kingdom.

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Study Suggests Genetics as a Cause, Not Just a Risk, for Some Alzheimer’s

People with two copies of the gene variant APOE4 are almost certain to get Alzheimer’s, say researchers, who proposed a framework under which such patients could be diagnosed years before symptoms.

By Pam Belluck

Scientists are proposing a new way of understanding the genetics of Alzheimer’s that would mean that up to a fifth of patients would be considered to have a genetically caused form of the disease.

Currently, the vast majority of Alzheimer’s cases do not have a clearly identified cause. The new designation, proposed in a study published Monday, could broaden the scope of efforts to develop treatments, including gene therapy, and affect the design of clinical trials.

It could also mean that hundreds of thousands of people in the United States alone could, if they chose, receive a diagnosis of Alzheimer’s before developing any symptoms of cognitive decline, although there currently are no treatments for people at that stage.

The new classification would make this type of Alzheimer’s one of the most common genetic disorders in the world, medical experts said.

“This reconceptualization that we’re proposing affects not a small minority of people,” said Dr. Juan Fortea, an author of the study and the director of the Sant Pau Memory Unit in Barcelona, Spain. “Sometimes we say that we don’t know the cause of Alzheimer’s disease,” but, he said, this would mean that about 15 to 20 percent of cases “can be tracked back to a cause, and the cause is in the genes.”

The idea involves a gene variant called APOE4. Scientists have long known that inheriting one copy of the variant increases the risk of developing Alzheimer’s, and that people with two copies, inherited from each parent, have vastly increased risk.

The new study , published in the journal Nature Medicine, analyzed data from over 500 people with two copies of APOE4, a significantly larger pool than in previous studies. The researchers found that almost all of those patients developed the biological pathology of Alzheimer’s, and the authors say that two copies of APOE4 should now be considered a cause of Alzheimer’s — not simply a risk factor.

The patients also developed Alzheimer’s pathology relatively young, the study found. By age 55, over 95 percent had biological markers associated with the disease. By 65, almost all had abnormal levels of a protein called amyloid that forms plaques in the brain, a hallmark of Alzheimer’s. And many started developing symptoms of cognitive decline at age 65, younger than most people without the APOE4 variant.

“The critical thing is that these individuals are often symptomatic 10 years earlier than other forms of Alzheimer’s disease,” said Dr. Reisa Sperling, a neurologist at Mass General Brigham in Boston and an author of the study.

She added, “By the time they are picked up and clinically diagnosed, because they’re often younger, they have more pathology.”

People with two copies, known as APOE4 homozygotes, make up 2 to 3 percent of the general population, but are an estimated 15 to 20 percent of people with Alzheimer’s dementia, experts said. People with one copy make up about 15 to 25 percent of the general population, and about 50 percent of Alzheimer’s dementia patients.

The most common variant is called APOE3, which seems to have a neutral effect on Alzheimer’s risk. About 75 percent of the general population has one copy of APOE3, and more than half of the general population has two copies.

Alzheimer’s experts not involved in the study said classifying the two-copy condition as genetically determined Alzheimer’s could have significant implications, including encouraging drug development beyond the field’s recent major focus on treatments that target and reduce amyloid.

Dr. Samuel Gandy, an Alzheimer’s researcher at Mount Sinai in New York, who was not involved in the study, said that patients with two copies of APOE4 faced much higher safety risks from anti-amyloid drugs.

When the Food and Drug Administration approved the anti-amyloid drug Leqembi last year, it required a black-box warning on the label saying that the medication can cause “serious and life-threatening events” such as swelling and bleeding in the brain, especially for people with two copies of APOE4. Some treatment centers decided not to offer Leqembi, an intravenous infusion, to such patients.

Dr. Gandy and other experts said that classifying these patients as having a distinct genetic form of Alzheimer’s would galvanize interest in developing drugs that are safe and effective for them and add urgency to current efforts to prevent cognitive decline in people who do not yet have symptoms.

“Rather than say we have nothing for you, let’s look for a trial,” Dr. Gandy said, adding that such patients should be included in trials at younger ages, given how early their pathology starts.

Besides trying to develop drugs, some researchers are exploring gene editing to transform APOE4 into a variant called APOE2, which appears to protect against Alzheimer’s. Another gene-therapy approach being studied involves injecting APOE2 into patients’ brains.

The new study had some limitations, including a lack of diversity that might make the findings less generalizable. Most patients in the study had European ancestry. While two copies of APOE4 also greatly increase Alzheimer’s risk in other ethnicities, the risk levels differ, said Dr. Michael Greicius, a neurologist at Stanford University School of Medicine who was not involved in the research.

“One important argument against their interpretation is that the risk of Alzheimer’s disease in APOE4 homozygotes varies substantially across different genetic ancestries,” said Dr. Greicius, who cowrote a study that found that white people with two copies of APOE4 had 13 times the risk of white people with two copies of APOE3, while Black people with two copies of APOE4 had 6.5 times the risk of Black people with two copies of APOE3.

“This has critical implications when counseling patients about their ancestry-informed genetic risk for Alzheimer’s disease,” he said, “and it also speaks to some yet-to-be-discovered genetics and biology that presumably drive this massive difference in risk.”

Under the current genetic understanding of Alzheimer’s, less than 2 percent of cases are considered genetically caused. Some of those patients inherited a mutation in one of three genes and can develop symptoms as early as their 30s or 40s. Others are people with Down syndrome, who have three copies of a chromosome containing a protein that often leads to what is called Down syndrome-associated Alzheimer’s disease .

Dr. Sperling said the genetic alterations in those cases are believed to fuel buildup of amyloid, while APOE4 is believed to interfere with clearing amyloid buildup.

Under the researchers’ proposal, having one copy of APOE4 would continue to be considered a risk factor, not enough to cause Alzheimer’s, Dr. Fortea said. It is unusual for diseases to follow that genetic pattern, called “semidominance,” with two copies of a variant causing the disease, but one copy only increasing risk, experts said.

The new recommendation will prompt questions about whether people should get tested to determine if they have the APOE4 variant.

Dr. Greicius said that until there were treatments for people with two copies of APOE4 or trials of therapies to prevent them from developing dementia, “My recommendation is if you don’t have symptoms, you should definitely not figure out your APOE status.”

He added, “It will only cause grief at this point.”

Finding ways to help these patients cannot come soon enough, Dr. Sperling said, adding, “These individuals are desperate, they’ve seen it in both of their parents often and really need therapies.”

Pam Belluck is a health and science reporter, covering a range of subjects, including reproductive health, long Covid, brain science, neurological disorders, mental health and genetics. More about Pam Belluck

The Fight Against Alzheimer’s Disease

Alzheimer’s is the most common form of dementia, but much remains unknown about this daunting disease..

How is Alzheimer’s diagnosed? What causes Alzheimer’s? We answered some common questions .

A study suggests that genetics can be a cause of Alzheimer’s , not just a risk, raising the prospect of diagnosis years before symptoms appear.

Determining whether someone has Alzheimer’s usually requires an extended diagnostic process . But new criteria could lead to a diagnosis on the basis of a simple blood test .

The F.D.A. has given full approval to the Alzheimer’s drug Leqembi. Here is what to know about i t.

Alzheimer’s can make communicating difficult. We asked experts for tips on how to talk to someone with the disease .