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On behalf of the authors, Brun Ulfhake sums up for us the recent paper on the use of DVC® System during the cage change.

Although alternatives are becoming more numerous and (are) validated, non-animal experiments can still not substitute all in vivo experiments. Studies that need to use live animals should be conducted deploying all possible refinements to minimize the harm inflicted and, furthermore, designed to produce conclusive results using the smallest number of animals possible.

Optimisation of husbandry routines is an important component of refinement and for laboratory rodents a cage-change that does not keep the cage interior at a high standard has proven to be one key element. Indeed, many studies have given evidence that the cage change is quite intrusive and stressful for the inhabitants, upsetting behaviour and sleep pattern, impacting the heart rate and blood pressure to name a few examples.

The impact of cage-change is a factor that clearly could impact the results of any study on small rodents. A good balance between leaving the animals undisturbed and an in-cage hygienic situation fitting the needs of the animals must be achieved.

Brun Ulfhake, MD, PhD, Senior Professor - Department of Laboratory Medicine at Karolinska Institutet, is one of the authors of the multicentre study on spontaneous in-cage activity and micro-environmental conditions of IVC housed C57BL/6J mice during consecutive cycles of bi-weekly cage-change.

Dear Brun, your article is important for the LAS community both for PIs and Facility Managers. Can you summarize it for our readers?

The reason for our study was to provide a more complete description of in-cage life and animal health during repeated bi-weekly cage changes.

We chose to study a mouse strain (C57BL/6J) which is very commonly used in life science research. By using the DVC® for housing, we could record in-cage rest and activity day and night across the cage-change interval. Furthermore, we used the DVC® technology to identify the position of the latrine(s) in the cages. The cages were custom adapted with small closable holes to enable measurement of ammonia across the full width of the rear middle and frontal sections of the cage floor. Thus, ammonia levels were collected during flow conditions and without having to remove the cage from the DVC®. Ammonia measurements were effected using an electrochemical detector technique at regular intervals (6-7 times) of each cage-change cycle. By the longitudinal gathering of in-cage activity, latrine positioning and ammonia levels, it was possible to compare the first and second week of the cage-change cycle. By analysing repeated biweekly cage change cycles, variations across cycles could be estimated. Another important feature of this study was that it was conducted in parallel at facilities in four different countries within the EU. This allowed us to identify observations that were common across sites from those that only showed at single sites. Finally, at one of the sites, the protocol was extended to include outcomes when housing density was changed from four to two animals, and when the bedding was changed from aspen chips to corn cob. Here we also measured in-cage bacterial load after bi-weekly and weekly cage-changes. At the end of the experiment, the upper airways of randomly selected mice were subjected to histopathological analysis.

DVC® has been an important tool in conducting the study and collecting such important data. Can you comment on it?

Importantly, in this study the DVC® technology enabled us to continuously monitor the home cage activity and rest of animals, without disturbing them. For us the spatial resolution provided by the twelve electrodes localized outside of the cage gave us the opportunity to analyse not only activity but also how the mice use the cage floor across cage-cycles. This is another important result of our study. Moreover, we used the drop in resistance due to wetting of the bedding to identify the location of the latrine inside the cage and relate this observation to the animals’ use of the cage floor.

What are the results of the study?

Our data show that cage change induces a marked increase in activity (~40%) being more pronounced during daytime when the animals normally rest than during nighttime. The subsequent decline from this activity burst occurred during the first week. Thus, the data strongly support the notion that from the animal’s perspective, bi-weekly cage change is to be preferred over weekly cage change.

Irrespective of the cage change frequency, the impact of a cage change is such that it must be incorporated into the experimental design as a variable. The histopathological examination of the nose cavity revealed mild to moderate signs of abnormalities that did not convey with the recorded in-cage ammonia levels. Seven out of the nine morphological signs were also present in the germ-free mice with no lifetime ammonia exposure suggesting that these may be caused by other in-cage components such as dust or chemicals from the bedding material. Further studies on bedding materials are needed.

A distinct improvement in in-cage microenvironment would be the development of a nontoxic and dust free material with properties that reduce the production of ammonia while meeting the demands of the mice.

Can you comment on the DVC® technology and tell us your vision of DVC® in the lab animal industry in the short term?

The DVC® technology is scalable using a standard IVC housing system. It is already used as a tool for facility management and emerging application may assist in notifying early-on a range of abnormal activities in the cage. The collection of data from the system does not call for an advanced digital infrastructure, such as massive data storage and processor power.

The 24/7 output of the system can be analysed in close-to-real time and provide unsupervised data on home-cage rest and activity, and with single housed animals also locomotion. Several more recent papers have shown that it is also a powerful tool in research of spontaneous 24/7 behaviours of small rodents and ideal for swift capturing of rhythmicities such as the circadian rhythm of day and night (PONE, 2019). We have used the system to study rhythmicities in home-cage activity induced by husbandry routines (PONE 2019).

Using cumulative records covering about 1.5 years, we were able to discover that laboratory mice show a slow rhythmicity (~3 months) in activity with a large effect size (≥0.7 SD) (Scientific Rep 2021). I would also like to mention that the system can be complemented with other features such as sound pickup, video capturing and more. It should be noted that such add-ons will increase the demand on the local digital infrastructure but feasible when only a smaller number of cages are equipped with add-ons. I am convinced that home-cage monitoring is the future in studies of the behaviours of small rodents.