Category: 3Rs

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“The business of animal suffering and death”

Last weekend I received the following e-mail:

The website of Lab-Animal-Training offers courses that train people to “carry out procedures on animals” and to “kill animals,” activities referred to on your website as “EU Function A” and “EU Function D.”

You call this “laboratory animal science,” but it is, in reality, the business of animal suffering and death. You profit from training people to ignore the most basic ethical principles.

The e-mail was sent to a number of recipients. For some reason, I and a colleague in Switzerland were singled out as individuals among a list of mainly organisations. Given the content, I assume that we were all chosen as having a role in training scientists for doing experiments with animals. The functions referred to are the ones of Article 34 in Directive 2010/63/EU which is the legislation regulating the use of animals in research in the EU, and one of the many things the Directive covers is the requirement for training before doing any work that involves animals.

I totally understand that it is disturbing that there are training courses with the focus on killing animals, in particular when it is as explicit as the Directive is about that this is precisely what it is. I appreciate that the Directive is not using euphemisms here; we all need to be aware of that what we are doing when we are ending the life of an animal is to kill it. And it doesn’t qualify for the term euthanasia unless it would be in the animal’s own interest and this is only the case for terminally ill animals for which nothing else can be done.

But there is a weird assumption of cause and effect. People are not doing experiments on animals because they have taken our courses – they take our courses because they are doing experiments on animals and therefore need the training our courses provide. It is not as if we’re luring people into doing experiments on animals by organizing training courses.

Are we teaching people to ignore the most basic ethical principles? It seems easy to assume that not to kill and not to do harm is one of the most basic ethical principles, and I respect people who decide to live fully according to that idea. It of course means not to consume products of animal origins, and for consistency many also argue that it must mean not to use any medical treatment that has been developed using animals. It is very obvious that society in general, in most places of the world, is not based on an ethical principle of not killing and not harming any living being.

What we do teach is how to do experiments on animals causing the least possible harm and suffering to them. This is what I call the compromise position, the one that legislation is based on and therefore somehow the official position in our society, the European Union.

The profit, then? Whether you call it surplus or profit, when income is greater than expenditure, you have money left after paying all the expenses, and you can do things with that money. For not-for-profit organizations, which is the category in which most course organizers are found, this money is used for other important activities within the organization. In our case for research, since that is our “core business”.

Thanks to the funds generated through training courses in laboratory animal science, we are able to invest in research that we believe in without having to wait for successful funding applications. So far, we have been able to fund a one-year fellowship for a young animal welfare scientist to do research into the welfare of shelter dogs that are being trained by prison inmates.

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Mice developing human cells – challenging how we think about ourselves or about animals?

This blog has been dormant for four years, and I don’t know if this will be a single post or actually represents a first step in attempting to re-establish a writing practice beyond papers and reviews and grant applications again. Time is an issue – not least because ideally I would like to research the topics I write about more than I have done this time. On the other hand, I’m hopeful that I’m on my way to recover some of my time and also inspiration, and arguably I also have more experience to fall back on wi

Two headlines in this week’s mail alert from Nature have inspired the heading.  Both have to do with animal embryos developing with human cells incorporated into their bodies. Japanese researchers have been successful in achieving this by injecting the cells directly into the amniotic fluid of pregnant mice, whereas a Chinese team reports growing hearts containing human cells in pig embryos. The Japanese project seems to be more of a technology breakthrough – if that term is even justified for what is perhaps better described as a successful attempt to do something the researchers themselves didn’t really expect to work:

The team used reprogrammed stem cells to grow human organoids of the gut, liver and brain in a dish. Shen says the researchers then injected the organoids into the amniotic fluid of female mice carrying early-stage embryos. “We didn’t even break the embryonic wall” to introduce the cells to the embryos, says Shen. The female mice carried the embryos to term.

“It’s a crazy experiment; I didn’t expect anything,” says Shen.

Within days of being injected into the mouse amniotic fluid, the human cells begin to infiltrate the growing embryos and multiply, but only in the organ they belonged to: gut organoids in the intestines; liver organoids in the liver; and cerebral organoids in the cortex region of the brain. One month after the mouse pups were born, the researchers found that roughly 10% of them contained human cells in their intestines — making up about 1% of intestinal cells

Of course, we should keep in mind that none of the studies have yet been peer reviewed and published. We don’t know the quality of the studies or whether the results can be replicated. But making animals that produce human cells has been a big goal for many researchers for decades, and I don’t see any reason why technology would not continue to develop in that direction. This is not to say that I don’t think this is an unquestionable given, but so far this research is ethically and legally possible in enough countries where there are also resources to develop it. And the technology hurdles are probably gradually being overcome – although as we will see below not everyone is convinced that this is happening any time soon.

Which, then, are the consequences seen from the perspective of my own area of research, animal ethics and the 3Rs of animal research?

In terms of the general ethics of how humans treat other animals, this represents another way in which humans use animals for their own benefit. Whether or not we agree with this widespread human attitude (and there’s a lot to be said about the ethical aspects of it!), for the animals the practice described here is not radically different from other ways they are handled in research and technology. Non-human animals are unlikely to suffer from the concept of being used; what matters is probably what they experience as a consequence of what is done to them – largely what we cover in considerations of the 3rd R of Refinement. Since the studies haven’t been published, how exactly the animals were handled is not known outside the research team itself, but the most affected animals are probably the females carrying the embryos, and that as a consequence of the procedures done to them rather than related to the nature of the embryos.  

The 2nd R – Reduction (of animal number) is where this kind of research really has consequences. On the one hand, given that the aim is to have liveborn animals with human tissue (for research or for transplantation), the use of animals is required (in other words, no possibility for Replacement, the first of the 3Rs). On the other hand, this is the kind of research that when successful may lead to an increasing use of animals. The possibility to do research on animals that have organs based on human cells is attractive for biomedical research as a way to overcome the biological differences between species, which then may increase the demand for research animals. In a paper entitled The 3Rs Alone Will Not Reduce Total Animal Experimentation Numbers: A Fundamental Misunderstanding in Need of Correction, philosopher Nico Müller at the University of Basel develops this argument:

The basic problem is that innovation occurs not just in NAMs, but also in animal methods. The assumption that NAMs innovation will in time lead to lower total distress discounts innovation at the other end of the spectrum, that of new distress-inducing animal methods. But of course, innovation in this area is constantly blooming, as every new animal experiment is also a methodological innovation. What is more, these innovations may often conform better to established paradigms and face fewer obstacles than innovative NAMs (Lohse 2021). A stark example of a breakthrough innovation in animal experimentation can be seen in the advent of genetically modified research animals, which led to a significant increase in total animal experimentation numbers worldwide (Ormandy, Schuppli, and Weary 2009). Actual conditions aside, in principle, for every distress-inducing technique replaced, reduced, or refined, two new ones can be invented, such that total distress increases despite the steady progress of the 3Rs. On the whole, the 3Rs can diminish total distress only under conditions that, by coincidence or by design, hinder the innovation and proliferation of new distress-inducing methods. But the 3Rs cannot ensure that such conditions obtain.

https://www.theguardian.com/science/2021/may/15/mixed-messages-is-research-into-human-animal-hybrids-ethical-chimera

Regarding the challenge to how we think about ourselves, I was about to say that this is a question that goes outside my own area of research. But that isn’t entirely true, in that we did discuss the question of species barriers in a series of interviews done by then PhD student Pedro Ramos and published two years ago in New Genetics and Society. We interviewed scientists working with gene editing, and their view of the issue is very similar to that of the scientists that spoke to The Guardian around the same time about chimeras:

Nakauchi, now at Stanford University in California, says that the ethics of such experiments are most fraught if they generate “ambiguous animals, such as a pig with a human face or human brain”. That might be impossible even in principle, given the evolutionary distance between pigs and humans – but for monkeys it’s less clear. So we should avoid making human-animal chimeras with a large human component, he says – and perhaps use genetically modified human stem cells that can’t make brain cells. Wu agrees, but stresses that they never intended to implant any of their chimeric embryos for further growth anyway – the aim is not to grow human organs in monkeys. He doesn’t think that should be contemplated if we don’t know where the human cells might end up. “What matters to me is where the human cells go and how many there are,” says biologist Marta Shahbazi, who works on embryo development at the University of Cambridge. “If we confine them exclusively to an organ of interest, like a pancreas, that’s OK: a mouse with a pancreas made of human cells is in no meaningful sense ‘human’. But for a mouse with human stem cells spread throughout all the tissues, the answer is not so clear. And for a monkey, things get even more blurry.”

What these scientists are speaking about is what philosopher Bernard Rollin describes as telos. As long as the pigness of the pig is not overrun by human characteristics that we prefer confined to the humanness of the human, things are fine. Of course, where to draw that line is not an easy task. Other scientists, however, have a different perspective altogether on whether this is something we need to discuss at all.

Alfonso Martinez Arias, a developmental biologist at Pompeu Fabra University in Barcelona thinks the claims of Izpisua Belmonte and colleagues are completely overblown anyway. He thinks all they have really shown is that when some human cells are added to monkey embryos, they become moribund. “If you add some extraneous cells that make it more difficult for the embryo to survive, what have you learned from what is a rather sick biological entity?” he asks.

“Human-monkey chimeras are not coming soon, and may never come,” he says. “In any case, we don’t need them – not to answer biological questions, nor to solve problems associated with organ donors.”

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Being open about animal research

Today is Being Open about Animal Research Day, #BOARD21. If I can pretend to speak on behalf of my institution, this is what I would say about us. This is what I’m trying to act in line with.

We do research with animals. We also develop non-animal models for biomedical research. Transparency means talking about both, in a way that is as unbiased and as honest as possible.

I’m not a typical researcher using animal models, and my way of being open about animal research will be particular for my own professional self and context.

I’m an animal welfare scientist in a biomedical research institution, i3s – Instituto de Investigação e Inovação em Saúde. I have worked in this organization for 20 years, I have learned a lot, unlearned some, and hopefully achieved one thing or another in terms of institutional culture, practice and infrastructure.

My greatest passion in the field is the research we develop to improve animal welfare, and in particular our research into survival of laboratory mouse pups. This is the topic I have worked on for the longest time, and it has brought fantastic collaborations with great colleagues and outstanding research institutions. At the point where we are now, I’m also believing our research finding can bring about change.

But changing the world through research is a very slow process. Training is much more impactful – or at least so I hope, since this is the other main part of my work. My institution has hosted a training course in laboratory animal science for researchers working with animals since 2005. It makes a huge difference for the mindset and for the standard that everyone who works with animals has been extensively trained to do so. It helps to create a spiral effect where knowledgeable researchers demand high quality support from the animal facility – and the animal facility can set high standards for how animals are treated in research. For us, adhering to international accreditation schemes – FELASA for our training course, AAALAC-International for the animal care and use program as a whole – has been crucial to keep up the quality and to improve.

Institutional measures to promote quality in research with animals are important. The same is true for quality in animal care. Working with international accreditation schemes (FELASA, AAALAC) help us to keep up the quality and improve.

But there is a lot more we can do.

I would like to know that all researchers when planning their research think carefully about what the right model is to address their research questions. And that they get support in this – that they are challenged to think, that they have experts to “think together” with, and that they get expert support in implementing the models. This should be the case for both animal and non-animal models.

I have a vision for how to achieve this within my own institution. To get the entire research community to think this way is beyond my reach, but of course it’s an important ideal.

In the meantime, I wish for all of us to be honest about what we do and what we can achieve. There are problems in practice with animal model research and problems with non-animal model research. We should work to prevent and overcome these problems, and not deny that they exist, or act as if they only happen in one type of research. There are limitations of animal models and limitations of non-animal models. If we speak about the limitations of one, we should also speak about the limitations of the other. Selective use of facts is not good practice in science communication. But it’s far too common in the discussion of animal experimentation – on both sides of the debate.

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Painting mice

Why are we recording a video of my colleague Rita Santos painting the back of a white IKEA mouse with a black marker pen?

The background story is that nearly 50 years ago, in 1974, a US researcher painted real laboratory mice as part of what has become a well known story of scientific misconduct in biomedical research. The story isn’t as well-known among biologists and animal scientists as it is among immunologists (or at least so I assume, based on anecdotal evidence = whom among my contacts and colleagues who were or were not aware of it when I asked), despite being both striking and somewhat sad.

Striking, because once you’ve heard the story, you are likely to remember it. Dr William T Summerlin was doing research into transplantation immunology. He believed that by keeping the tissue in laboratory culture for some time before transplanting it into the recipient animal, it could be transplanted without rejection. His proof-of-concept experiment was to graft skin from black mice to (genetically unrelated) white mice. Did he actually ever transplant skin? I don’t know – this amount of detail is not given in the easily accessible internet sources. But what is very clear is that his demonstration of success was a fraud. An attentive technician discovered that the black patch on the back of the white mouse could be rubbed off with ethanol. As reported in this NY Times account of the case, published only a month later, Summerlin admitted to having painted the mice.

Sad, for a number of reasons, going beyond the actual misconduct itself, which is of course in itself highly lamentable. The painted mice seems not to be a one-off event – when Summerlin was investigated the committee also discovered a seemingly very dubious case of cornea transplant experiments in rabbits. Whereas having their backs painted would hardly have harmed the mice, the failed cornea transplants must have caused the rabbits pain. And none of this was justified. Scientific experimentation is not about simply trying something to see if it works: there has to be a reasonably developed idea of what mechanisms are involved. I don’t find any reference to a theory about mechanisms involved in the purported transformation of a xenograft (from a genetically different individual) into tissue that is not recognised as foreign when transplanted into a recipient. All that is to be found is that Summerlin had claimed for some years that he had a method for laboratory culture of tissues that removed the problem with transplant rejection, and that other researchers were unable to make the procedure work when they tried to repeat it in their own labs (a classic way through which fraudulent or poorly conducted research is discovered). The requirement that an experiment is based on a reasonably developed theoretical framework and previous, related studies is even stronger when the health and well-being of living beings are involved.

Why, then, are we painting mice? As part of the INTEGRITY project, we are developing teaching material into ethics and research integrity issues in animal experimentation, for high school students, ready for road testing in about a month. And please note, we’re not painting mice, we’re painting toy mice. The first R, Replacement, of the 3Rs principle of course. Knowing what mice are like, I actually believe that using real mice for this purpose would not only have been stressful for them but a pain for us!

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Of hens, mites and teabags: Interview with Francesca Nunn

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Congratulations Francesca Nunn to the NC3Rs Prize! You were awarded this prize for your work on a new approach to testing treatments against red poultry mites. Tell us more about the project!

Poultry red mites are parasites that live in hen houses and emerge at night to bite hens and consume blood. There are a number of studies ongoing worldwide to develop new ways of controlling the mites but this is a really tricky host:parasite model to work with because the mites spend most of their time off of the host. To help with this, we needed a technology which kept small numbers of mites on the host and allowed feeding and recovery of the parasites. The NC3Rs funded project was to optimise and further develop a prototype “on-hen feeding device” that had achieved ~50% mite feeding in a pilot study. This device allows accurate assessment of mite control methods on small numbers of hens before conducting field studies, for example. This addresses “Reduction” by greatly reducing the number of hens used, as it would accurately identify poorly performing mite control methods before they were progressed to field trials. This system can also be used to test the effectiveness of mite control methods across prolonged periods on small numbers of hens (4 per treatment group, as opposed to 400 per treatment group in field trials) without continually exposing birds to the parasites. This therefore addresses “Refinement” as it allows the birds to remain free from the parasites, with parasites only accessing the birds for short (3 hour) periods every 3 weeks instead of the continual exposure encountered in field trials. The project involved developing the device for all the blood feeding mite stages as well as studies to optimising feeding rates, minimising background mite mortality and using the device in trials to test its performance.

Your approach allows a huge reduction in animal numbers. What about animal welfare? What would a traditional test approach be like for a hen, and what will she experience with your refined method?

Novel systemic acarines or vaccines are tested on hens using an experimental infestation model. This involves releasing a set number of mites into a cage of hens, and then monitoring the mite population growth over time. And of course, you’d compare the treatment group to a control group. This means that the experimental hens are exposed to thousands (often tens of thousands) of mites over a number of weeks. As we know, mites cause discomfort and stress to the hens which is why we need the treatment in the first place! Using our feeding device, the hens are only exposed to 50-100 mites per time point – the rest of the time they are free to just be hens in enriched floor pens and parasite free.

Can you tell us something about where the idea came from and how you went from idea to device?

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Similar rigid devices, which were glued onto the hen had been used previously but weren’t widely adopted. This is probably due to the mites not being able to attach to the hen when the hen’s movement caused the device to move. The team at Moredun, led by Dr. Kath Bartley and Dr. Al Nisbet, came up with a tea bag type prototype which solved this problem while also managing to contain the mites. The next issue was to find a material that the mites could feed through-originally we used phytoplankton mesh but had to find an alternative that allowed the much smaller nymph stages to feed also without escaping.