The 3Hs Quiz serves as a pivotal assessment within the course dedicated to enhancing rodent welfare in laboratory settings. It encompasses three fundamental aspects – Housing, Handling, and Habituation (the 3Hs) – crucial for promoting the well-being of research rodents.

Through questions addressing optimal housing conditions, proper handling techniques to minimise stress, and strategies for habituating rodents to their environment and protocols, the quiz evaluates practitioner's comprehension and application of essential principles. From understanding the importance of cage enrichment and cleanliness to mastering gentle handling practices and promoting behavioural flexibility through habituation, the quiz aims to equip technicians with the knowledge and skills necessary to create environments where rodents can thrive while ensuring the integrity and reliability of scientific research.

Complete The 3Hs CPD e-course and receive a certificate demonstrating your understanding of best practise in rodent housing, handling and habituation.

There are lots of different ways to provide enrichment for laboratory rodents. The most common approach to this is to provide resources within the cage which the animals are thought to value e.g. nesting material, shelters, conspecifics. 

Research suggests that enrichment has positive impacts for animal welfare but it is important to consider the specific needs of the species. Our understanding of the best ways to provide enrichment are also changing all the time as we do more research and apply objective methods to quantify the impacts. 

Mice – commonly thought to be highly social, fighting within groups of male mice is well known and can cause significant welfare issues.  

Mice – benefit from shelter and nesting material. Mice particularly value nesting material in laboratory environments where the temperature in most facilities is much colder than in their native habitat. Group housing may also help with thermoregulation in female mice and young males (before sexual maturity). 

Mice – prone to stereotypic behaviours which can include excessive use of resources such as wheel running 

Rats – good evidence for high levels of sociability and benefits of social contact. 

Rats – intelligent and benefit from resources which they can interact with e.g. chew blocks, forage mix, play pens 

Play pens – space restrictions as well as scientific objectives can limit the provision of enrichment within the home cage environment however, short periods of access to play pens can provide welfare benefits without having to make major changes to the standard housing provision. 

Enrichment for laboratory rodents can include providing animals with access to other conspecifics (social enrichment) and/or access to resources which they value (environmental enrichment).

The lifetime experience of the animal is key to understanding the welfare costs and for laboratory animals this is from when they are born to when they are killed. Provision of enrichment is known to benefit welfare and so this should be provided throughout the animal’s life even if their purpose is to be used in short term studies or for tissue collection. 

Enrichment does not benefit animals which are only being used for short term studies or for tissue collection.

It is important that approaches to enrichment are compatible with the scientific objectives otherwise they are unlikely to be widely implemented. 

Finding the optimal levels of enrichment relative to the scientific and facility needs is key to getting widespread uptake and maximise the numbers of animals which benefit.  

Too much enrichment within a cage can impact on how readily animals are health checked etc and this may impact on their welfare from a different perspective. 

Although environmental enrichment has positive welfare benefits in most circumstances, there are some situations where providing additional resources can lead to conflict within the cage and add to social stress e.g. group housed male mice may fight with each other if there are highly valued resources but this may occur less in barren cages.  

As more is understood about the affective state of animals under these different scenarios, it should become possible to make more objective recommendations. 

The more enrichment you can provide the better for the animal’s welfare.

There may be certain types of experiment where providing animals with enrichment can impact on the results of the experiment for example, changing the phenotype of certain GAA mouse lines, reducing animals’ likelihood to develop drug self-administration. The potential to cause changes to the scientific outcome is not a reason not to use enrichment but it can be pertinent to test a cohort with and without enrichment to establish the impact on the scientific readout(s). Something else to consider is the validity of the model more generally if enrichment has a major impact and implications of this more generally.  Providing laboratory rodents with enrichment will impact on their development and physiology but may actually provide a more ‘normal’ animal and therefore bring scientific benefits. As scientists we sometimes forget about the wider aims of our work and ultimately even fundamental biology research needs to be relevant to ‘normal’ humans or animals which current laboratory environments may not adequately reflect.

Environmental enrichment is detrimental for scientific outcomes.

Certainly for rats (and maybe for mice), even short periods of access to enriched environments can benefit their welfare. This approach may be particularly helpful when managing long term studies and may even provide a better overall model by reducing the development of negative affective states, obesity and stereotypic behaviours. This type of management is still in its infancy and so further research is still needed to fully understand the best approaches to use.  

For rats doing long term behavioural studies, access to the play pen can be helpful when animals are not needed for a period of time e.g. for drug washout or their experimenter is on annual leave. 

Using play pens is also beneficial for staff wellbeing where providing positive experiences for animals in their care is a rewarding experience. 

Access to play pens even once per week can benefit animal welfare.

All animals (including humans) find physical restraint aversive and it can generate fear, increase stress hormones and the effects accumulate with repeated experiences. Studies quantifying faecal pellets, plasma corticosterone and affective state all provide evidence of the negative welfare impacts of physical restraint.

Restraint in rodents is associated with distress.

Although this is the ‘normal’ response seen in mice and rats within the laboratory environment, this is more a reflection of how most are managed. In animals which have undergone habituation to handling using positive reinforcement, production of urine and faecal pellets during routine handling is actually quite unusual. 

Production of urine and faecal pellets in response to human handling is normal and not a sign of distress.

When animals are physically restrained this leads to distress and struggling behaviour which can then result in animals being injured during a procedure or causing them to bite the handler. When animals are managed without physical restraint or using methods which they have been trained for, they are less likely to bite the handler (particularly in the case of rats). Even if the animal does try to bite, it will look to address the cause of the discomfort e.g. the needle and not the human handler. 

A surprisingly common misconception is that animals must be inverted to ‘move the intestines’ for intraperitoneal injection. There is no evidence to support this and in fact, firm restraint methods may even make it more likely that a needle enters part of the GI tract that the modified method illustrated here where the abdomen is relaxed.

Animals must be physically restrained to ensure both their safety and that of the handler.

There is a growing body of literature which supports the welfare benefits of handling methods which avoid physical restraint including methods which involve the use of the tail.

Even in strains of mice which can be particularly difficult to handle, use of non-aversive methods such as tube handling should be used where possible. Taking time to habituate animals may also have scientific benefits by reducing the variability of data. Whilst some researchers and animal management staff have raised concerns about the time involved in using non-aversive methods versus the welfare benefits, the 3Rs framework puts an expectation to use the most refined methods and this includes for routine care and management, not just scientific procedures. 

Non-aversive handling methods, including avoiding method which involve tail handling, benefit animal welfare.

Self-ingestion of substances in palatable solutions can provide a simple and effective method which benefits animal welfare. In most cases, oral gavage requires physical restraint and therefore carries a higher welfare cost than other methods of substance administration which can be performed using refined handling methods. A variety of palatable vehicles are readily self-ingested by rodents. Not all drugs can be administered this way and a pilot study may be needed but once a protocol and vehicle have been established, all future treatments can adopt these refined methods.

Rats and mice will learn to ingest substances in palatable solutions avoiding the need for restrained methods such as oral gavage or injections.

Some researchers are concerned about the calorific value of palatable solutions. There are also some drugs which have a very bitter taste and that can affect animals ingesting them. One option is to use an artificial sweetener either as the vehicle alone or in combination with one of the other vehicles.

Adding sweetener can help counter the bitter taste of a drug.

Rodents are usually very cautious about ingesting novel foods and so are unlikely to ingest a substance in a palatable solution if they have not first been trained to ingest the vehicle. Using the vehicle as the positive reinforcer during initial habituation to handling is a useful way to prepare animals for both handling and substance administration at the same time. 

Providing animals with access to palatable foods before training for handling or substance administration is important to overcome neophobia.

Definitions for these terms do vary but in general, habituation in animal research relates to a method which is designed to train an animal for handling and/or a procedure such that the procedure does not in itself cause distress. This is best achieved using positive reinforcement.

Habituation and acclimatisation are the same thing.

There are many reasons why an animal can stop reacting when exposed to a procedure, including handling, but not all are associated with habituation and positive welfare benefits. Many scientists have misunderstood the differences between habituation, acclimatisation and learned helplessness and assume that they are habituated when they stop struggling or vocalising.

You know an animal has habituated to a technique when it stops struggling and/or vocalising.

To reduce animals experiencing distress, the process of habituation should be gradual. For most laboratory rodents, their initial contact with humans will be stressful and this cannot be avoided. Rodents are prey animals and they need to learn that human interactions do not mean harm but initially they will experience a fear response. To minimise this and maximise the positive experience, the first stages of handling should be very brief and positive reward provided. Once the animals associate human contact with reward they will start to see humans and anticipate reward which in itself generates a positive affective state. The duration of contact and what is expected of the animal can also be gradually increased until they achieve the outcome required.

Habituation protocols should gradually introduce the animal to the procedure using incremental steps which avoid animals experiencing anything aversive.

Conditioned aversion develops when an animal forms a negative association with an environment or cue e.g. being picked up by a human. Just as seen in the fear conditioning model, the animal then has to extinguish this memory and so habituation can take longer in animals which have already formed a negative association with human interaction. Unfortunately, this is probably quite common for laboratory animals as suppliers and breeding establishments do not routinely habituate animals to handling, use positive reinforcement or refined handling methods.

Conditioned aversion is more difficult to overcome than training a naïve animal.

Habituation protocols should introduce animals gradually to what is required including for basic handling. This helps to reduce animals experience of fear and aversion and in the long run, makes for a better and quicker habituation. Protocols should also consider the individual animal and adapt based on their behaviour. For example, some strains of mice can be much more sensitive to initial contact with humans and may require longer to habituate.  

Habituation is about training animals for the procedure you need them for and few scientific procedures will involve 10min of ‘stroking’ and animal. Most studies involve either: 

Transferring the animal from the home cage to a testing location

Transferring to induction chamber for anaesthesia 

Therefore, a habituation protocol should aim to build towards this over a few days using positive reinforcement at each stage to associate contact with the handler with a positive outcome. 

Stroking a mouse or rat for 10min each day for several days is a good habituation protocol.

Animals produce faecal pellets when they experience fear. Despite what most animal researchers think, production of urine and faecal pellets in response to human handling is not a given and in well handled animals which have been habituated using positive reinforcement few if any will produce faecal pellets or urinate in response to the standard handling methods described in the 3Hs.

Faecal pellets are a good way to gauge how effective a habituation protocol is.

For an animal to form a positive association with human handling, the reinforcement needs to occur within a specific timeframe. This is easiest to achieve by giving the reward immediately after the interaction. 

The time the positive reinforcement is given to the animal is not important.

Although we don’t know for sure, based on current evidence, mice are not as sociable as previously thought, and of this list probably would place most value on the nesting/bedding material. The laboratory environment is usually kept at ~20°C and for mice, this is >5°C below there thermoneutral zone i.e. the temperature which they are best suited for from a physiological perspective. 

To which of the following resources will mice attribute the most value?

Although we don’t know the answer based on current studies, we know rats are highly social and therefore are likely to value contact with other rats. They are better suited to the laboratory environment in terms of temperature and do not tend to use nesting/bedding material unless preparing for a litter or caring for young. Females are more likely to nest build than males. Rats do value shelter as they are prey animals and also benefit from objects they can manipulate such as chew blocks. Studies which have specifically compared these different resources have not been carried out so we can’t be sure of the answer.

To which of the following resources will rats attribute the most value? 

Both rats and mice can be trained to ingest test substances in a solution from a syringe, in a paste or even specially made biscuit or jelly. Some palatable solutions may be more suitable for the test substance you are using versus others e.g. milk proteins may impact on absorption so fruit juice may work better. Drugs which are not soluble in aqueous solutions can be mixed with nut oil and added to peanut butter for example. The concentration of the solution can also be adjusted to help mask the taste of bitter drugs. See our guidance document for more information and this is also regularly updated as we learn more about the best vehicles and how to work with some of the more challenging substances e.g. those which are bitter, cause nausea.

Which of the following vehicles can be used to administer test substances?

There are well established welfare reasons to avoid physical restraint in rodents because of the distress it can cause however, it can also have major impacts on the scientific outcomes, something which is often not given as much consideration.  Animals respond differently to restraint and stress which can increase the variation of the data, can change the phenotype and response to manipulations. Because the levels of stress experienced by the animal can vary between handlers and establishments, this can also impact on the reproducibility of results.

What are the welfare and scientific costs associated with physical restraint in rodents?

Considering the methods for substance administration described in this course, rank the following routes of administration from most refined to least refined:

Oral self-administration is the most refined method as the animal is voluntarily ingesting the palatable solution containing the substance. Using refined handling methods, SC and IP are also less stressful that conventional restrained methods but will still involve some discomfort due to the injection. If the substance could not be administered in a palatable solution, then SC using refined handling methods would be associated with the least suffering. When planning experiments, it is helpful to always consider the route of administration particularly for substances routinely used by the research group. There is a growing interest in this aspect of animal research in general and so keeping up to date with the literature is a good way to hear about new advances.

Considering the methods for substance administration described in this course, rank the following routes of administration from most refined to least refined when using oral gavage with physical restraint:

The principles of the 3Rs means that researchers must use the most refined methods to achieve their scientific objectives and so continuing to use a less refined approach is not often justified. Many scientists are concerned that changing any aspect of the methods could lead to issues with replicating previous findings. This can be easily addressed using a pilot study to directly compare the refined methods with previous methods used in the model. Although this will involve a small number of additional animals, ethically it is better for the animals overall i.e. refined methods can be used going forward with positive impacts on many more animals.

It is often difficult to decide when to change methods as there is always ongoing studies and so waiting until the end of a programme of work or changing to a new model can seem logical but in reality, often means the new methods can pushed back or never adopted. There is evidence that animals which are raised in more natural environments, and which are managed to achieve positive welfare can respond differently in certain tests or disease models. Some argue that this is a justification for not changing to higher welfare standards, but the counterargument is that abnormal animals e.g. a chronically stressed rodent housed in conditions which fail to meet is basic welfare needs, is a poor model for human biology and to study disease pathways or novel treatments.  

Your supervisor is concerned that changing methods could impact on the findings from your animal models. What is the best way to address this in your future study design?

The 3Hs CPD Course