Voluntary Group-Housing Compatible Fecal Sample Collection in Mice Using a Custom-Made Cage System

Summary

This protocol describes a custom cage system for voluntary, welfare-friendly, simultaneous, and individual fecal sample collection from group-housed mice.

Abstract

Reliable measurement of fecal corticosterone metabolites requires fecal samples that can be assigned to individual animals and collected within a defined time window. This is challenging in group-housed mice, where pooled home-cage samples do not allow individual assignment, and temporary separation may interfere with animal welfare and study outcomes. The goal of this protocol is to present a simple, inexpensive cage system that enables voluntary, simultaneous, and individual fecal sample collection from group-housed mice. The system consists of a wire-topped, polycarbonate home cage connected to three individual collection boxes. After habituation and training, mice voluntarily enter the boxes to access a small food reward. The doors can then be closed without chasing, handling, or forced transfer. Fecal pellets are collected from each box separately, allowing samples to be assigned to individual animals while maintaining social housing outside the sampling period. This protocol supports non-invasive longitudinal sampling and is suitable for studies measuring fecal corticosterone metabolites or other fecal biomarkers. The design is easy to build from standard cage material and acrylic glass, can be disassembled for cleaning, and allows several cages to be sampled in parallel. By reducing handling-related disturbance and improving standardization of the sampling window, the method provides a practical refinement for endocrine monitoring in group-housed mice.

Introduction

A major challenge in assessing plasma corticosterone concentrations is that the sampling procedure itself can induce an acute corticosterone increase, thereby confounding the measurement and potentially affecting the interpretation of the study results^1,2,3. Measuring fecal corticosterone metabolites (FCMs) provides a non-invasive alternative, as FCM concentrations reflect circulating glucocorticoid levels several hours before sampling, depending on species, strain, sex, and gut passage time^4,5. However, reliable FCM analysis in group-housed mice remains challenging. Pooled fecal samples collected from the home cage cannot be assigned to individual animals and may show high variability due to inter-individual differences in glucocorticoid secretion and uncertainty in the exact sampling window. Separating animals for extended periods may interfere with study objectives, while short-term separation for individual sample collection is labor-intensive, limits throughput, and may itself induce stress².

To enable individual fecal sample collection from group-housed mice with minimal handling, a custom-made fecal collection cage was developed (Figure 1A-E). The design was adapted from a previously described fecal collection cage for spiny mice⁶ and modified for use with laboratory mice. The system consists of a standard polycarbonate type III cage connected to three individual collection boxes, which the animals can access voluntarily and close using sliding doors. This setup allows fecal samples to be collected from individual mice without direct handling during the sampling period and enables simultaneous sampling of multiple animals. The cage, therefore, provides a practical approach for non-invasive, individually attributable FCM sampling in group-housed mice.

The following protocol describes a fecal sampling system that allows the collection and identification of fecal samples from individual mice while maintaining a group-housing environment, using custom-made cages for contactless feces sampling.

Protocol

The responsible animal ethics committee of the Medical University of Vienna was consulted before the study was conducted and advised that no formal animal experiment license was required; therefore, no approval or protocol number is available. The study involved established breeding and husbandry practices only, together with non-invasive fecal sample collection. No invasive procedures were performed. In accordance with Directive 2010/63/EU, the expected burden to the animals was assessed as being below the threshold of pain, suffering, distress, or lasting harm equivalent to or higher than that caused by the introduction of a needle in accordance with good veterinary practice. All animals were housed and cared for according to institutional animal welfare standards.

1. Building of the cages (see material list)

1. Cut parts 1-4 from 5 mm Plexiglass/acrylic glass according to the templates shown in Figure 2A-C.
   NOTE: Templates with exact dimensions can be printed in DIN A3 format (Supplementary Figure 1 and Supplementary Figure 2).
2. Cut off the bottom of the polycarbonate type II (PTII) cage as indicated in Figure 2D. This part will later serve as the top cover of the fecal collection boxes.
3. Drill the holes and slots into the former bottom of the PTII cage as indicated in Figure 2D.
   NOTE: Exact dimensions are provided in Supplementary Figure 3.
4. Drill holes into the front side and short side of the PTIII cage as indicated in Figure 2E.
   NOTE: Exact dimensions are provided in Supplementary Figure 4.
5. Use part 2 as a template to mark the corresponding drill holes on the short side of the PTIII cage and on the long side of the PTII cage.
6. Drill the marked holes and attach the PTIII cage, the PTII cage, and part 2 using the threaded screws, as shown in Figure 2F.
7. Insert part 1 as the bottom of the PTII cage. Position parts 4a and 4b so that they hold part 1 in place and divide the sampling cage into separate collection boxes. Use the hot glue gun to fix them in place.
8. Slide the lower protrusions of parts 4a and 4b into the slots of part 1. These protrusions form the legs of the collection boxes.
9. Slide the upper protrusions of parts 4a and 4b through the top cover, the former bottom of the PTII cage, to hold it in place.
10. Thread the wooden chopsticks or rods through the holes in the protrusions, using one rod at the top and two rods at the bottom, to stabilize and fix the construction.
    NOTE: Wooden materials should only be used in the vivarium if they are disposable, as they are not cleanable. If this is not the case, rods of alternative composition should be used.
11. Close remaining gaps or minor inaccuracies using a hot glue gun. Use parts 3a, 3b, and 3c (Figure 2B) as sliding doors between the home cage and the collecting boxes.
    NOTE: Ensure that no sharp edges, glue residues, or protruding material remain accessible to the animals. The acrylic glass/Plexiglass components used in this prototype are not suitable for steam autoclave sterilization. Therefore, if the system is used in SPF facilities requiring sterilized cage-related material, either an institutionally approved non-autoclave disinfection procedure should be used, or the acrylic components should be replaced by autoclavable material while retaining the same dimensions.

2. Animal preparation and housing

1. Obtain the required number of mice of the appropriate strain(s) for the study.
   NOTE: The representative data were obtained from 30 eight-week-old C57BL/6J mice comprising 20 females and 10 males. The mice were specific pathogen-free according to FELASA standards and were obtained from Charles River Laboratories, Sulzfeld, Germany.
2. House animals under conventional conditions in adapted polycarbonate type III cages with a maximum of three adult mice per cage.
   NOTE: Separated males were housed in standard polycarbonate type II cages for this study.
3. Maintain the animal room at 22 ± 2 °C and 55 ± 10% relative humidity under a 12 h light/dark cycle with lights on at 6:00 a.m.
4. Provide a standard maintenance diet and water ad libitum.
5. Add aspen wood bedding, a paper toilet roll, a red transparent plastic house, and paper towels as nesting material to each cage.
6. Identify mice primarily by natural tail markings. Temporarily apply pen markings to animals with similar tail markings during weighing, if required.

3. Habituation and training of the animals

NOTE: The completed cage consists of a home cage, PTIII, connected to a sampling cage with three separate collection boxes, PTII. The home cages can be used like conventional cages, with bedding, cage enrichment, a wire lid without external fasteners, and providing food and water. The system can be disassembled for washing and can be used only during sampling weeks. During weeks without sampling, animals can be housed in standard PTIII cages.

1. Place the animals in the cage system at least two weeks before the first planned sampling session.
2. Habituate the animals to the collection boxes by removing the sliding doors for one night, allowing the animals to freely explore the boxes.
3. Perform three training sessions before the first sampling session. Conduct training under the same light conditions and at the same time of day planned for later sampling, ideally under red light shortly after the beginning of the dark phase.
4. Apply a small amount of reward, such as hazelnut cream, peanut butter, malt paste, or a similar palatable food, to the wall opposite the entrance of each collection box.
5. On the first training day, leave the doors open and allow the animals to discover and consume the reward voluntarily.
6. On the second training day, wait until an animal enters a collection box and starts licking the reward. Carefully close the sliding door behind the animal and release it after 10 min.
7. On the third training day, repeat the procedure and gradually extend the time the animals remain in the collection boxes.
   NOTE: Training should be calm and consistent. Avoid chasing the animals into the boxes, as this may increase stress and reduce voluntary participation in later sampling sessions.

4. Collection of fecal samples

1. Timing of sample collection
   1. Plan the sampling time according to the biological question and the expected delay between plasma corticosterone and fecal corticosterone metabolite excretion. In mice, fecal corticosterone metabolites typically reflect circulating glucocorticoid levels approximately 8-10 h before sampling.
      NOTE: If precise timing is critical, perform a pilot study to determine the time course of FCM excretion in the specific strain, sex, and experimental conditions used. This pilot can be combined with the training sessions.
   2. Schedule sampling during a period of high fecal output. Sampling is usually most efficient at the beginning of the dark and active phase.
   3. Perform all sampling sessions at the same time of day throughout the study to reduce variation caused by the diurnal rhythm of FCM levels.
      NOTE: In our experience, events occurring in the morning around 10:00 a.m. are well reflected when sampling is scheduled between 6:00 p.m. and 8:00 p.m., depending on the light schedule of the animal room.
2. Sampling procedure and practical advice
   1. Mark the animals individually before sampling. Use ear marks or individual tail markings.
      NOTE: Toe markings and color codes may be difficult to recognize inside the boxes under red light.
   2. Prepare the collection boxes during the light phase by applying the reward to the back wall of each box.
      NOTE: Preparing the boxes during the day improves visibility for the experimenter and allows the animals to smell the reward before sampling.
   3. At the planned sampling time, open the collection boxes and wait until the animals enter voluntarily.
   4. Once a mouse has entered a collection box, carefully close the sliding door behind it.
   5. After all animals have entered a box, record the identity and position of each mouse in a prepared sampling sheet.
   6. If more than 10 cages are sampled in one session, involve a second experimenter to support animal identification, door handling, and documentation.
   7. Keep the animals in the collection boxes for the planned sampling duration, typically 30 min to 2 h, depending on the expected fecal output and study design.
      NOTE: Exclude samples if individual identity cannot be assigned with certainty, if fecal pellets are contaminated with urine, bedding, or feces from another animal, if the sampling duration deviates substantially from the planned time window, or if the amount of fecal material is insufficient for FCM analysis.
      NOTE: The use of 50 mg is our standard protocol, and this quantity has been used in all the validations and all other papers^5,7,8. Normally, it can be easily obtained.
   8. At the end of the sampling period, open the sliding doors and allow the mice to return to the home cage.
   9. Close the collection boxes immediately after the mice have left to prevent re-entry and cross-contamination of samples.
   10. Remove fecal pellets from each collection box using long forceps and transfer them into individually labeled collection tubes.
   11. Store samples at -20 °C until further preparation of the FCM assay.
       NOTE: If the boxes are securely closed and cannot be re-entered by the mice, fecal pellet collection can be postponed until the following light phase. However, this should be done consistently within a study.
   12. Keep the animals in the home cage part (PTIII) until the next cage change. Then they can be disassembled and washed in a usual cage washer.

Results

Fecal corticosterone metabolites (FCMs) were successfully quantified from samples collected with the custom-made fecal sampling cages in a longitudinal study comparing permanently and intermittently bred mice. The representative FCM analysis included 30 animals: permanently bred group, n = 15 animals in 5 cages; intermittently bred group, n = 10 animals in 5 cages, with 5 males single housed except for 3 breeding events, where they were co-housed with the females. Samples were collected at 19 biweekly time points on days...

Discussion

This protocol describes a simple cage system for collecting individually assignable fecal samples from group-housed mice. The main advantage of the method is that it combines individual sample attribution with the maintenance of key aspects of social housing, particularly visual and olfactory contact with cage-mates. This is relevant because group housing is often required for animal welfare and may also be part of the experimental design¹⁰. In contrast to pooled home-cage sampling, the system all...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Drill bit 5 mm	Craftomat	22380487	5mm diameter; Quantity 1
Drill bits 60 mm	Bosch	32064647	60 mm diameter; Quantity 1
Drill bits 7 mm	Craftomat	22380520	7 mm diameter; Quantity 1
Drilling machine	Makita	25730054	Standard handheld or bench drill; Quantity 1
Hot glue gun	Einhell	29051207	For cage assembly; Quantity 1
Hot glue sticks	Einhell	29049664	Compatible with hot glue gun; Quantity 5-10
Makrolon cage	Animalab	31200129	Standard Type II cage; Quantity 1
Makrolon cage	Animalab	31200256	Standard Type III cage; Quantity 1
Plexiglass / acrylic glass	Bauhaus	29122352	Approx. 0.25 m² total; 5 mm thickness; Quantity  2DIN A3 sheets
Saw or milling device	Scheppach	32079641	Small band saw, jigsaw, or CNC mill; Quantity 1
Threaded screws	Profi Depot	10807533	20 mm × 3 mm; Quantity 4
Wooden chopsticks / rods	Ikea	903.429.71	Round wooden rods; Quantity 3