In 2010 the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were set up to improve reporting of animal research. In 2020 an update of the guidelines was published:

https://doi.org/10.1371/journal.pbio.3000410

The reorganised guidelines were presented in 2 sets, ”The ARRIVE Essential 10” and ”The Recommended Set“.

Here we show the realisation of the ARRIVE guidelines for the GMC.

 

 

 

The ARRIVE Essential 10

These items are the basic minimum to be included in a manuscript. Without this information, readers and reviewers cannot assess the reliability of the findings.

 

 

1. Study Design

Recommendation

For each experiment, provide brief details of study design including:

a. The groups being compared, including control groups. If no control group has been used, the rationale should be stated.

b. The experimental unit (e.g. a single animal, litter, or cage of animals).

GMC Approach

a. We compare a mutant group with the respective control group for each sex (female and male): The control group consists of littermates of the experimental group.

b. The individual mouse was considered the experimental unit within the project.

 

2. Sample Size

Recommendation

a. Specify the exact number of experimental units allocated to each group, and the total number in each experiment. Also indicate the total number of animals used.

b. Explain how the sample size was decided. Provide details of any a priori sample size calculation, if done.

GMC Approach

a) We used a sample size of n=15 mice per genotype and sex. Consequently, the total number of mice used in one experiment is N=60. Reasons for deviations of this number (e.g. dead animals) are documented.

b) The selected sample size of 15 mice per sex and genotype is sufficient to find a medium difference of one standard deviation with a power of 0.8 and alpha of 0.05. For some parameters (e.g. categorical, yes/no) even less animals are sufficient.

Sample size estimation is based on long-term experience in high-throughput phenotyping. Since we conduct a high throughput screening we apply no a priory sample size calculations in the format of power considerations.

In the sense of 3R, we do not include reserve animals. The estimated sample size is sufficient to generate a robust data basis, even in the rare case of shortfall. Further reserve animals are not applicable, since it is strongly recommended to only use animals with the same handling during the screen, to ensure comparability.

 

3. Inclusion and Exclusion Criteria

Recommendation

a. Describe any criteria used for including and excluding animals (or experimental units) during the experiment, and data points during the analysis. Specify if these criteria were established a priori. If no criteria were set, state this explicitly.

b. For each experimental group, report any animals, experimental units or data points not included in the analysis and explain why. If there were no exclusions, state so.

c. For each analysis, report the exact value of n in each experimental group.

GMC Approach

a. In general, all mice with genotypes that are to be compared  (e.g. homozygous versus wild type control animals) that result

from for the purpose appropriate breedings (e.g. heterozygous x heterozygous to generate homozygous and wild type animals) are considered to reflect a representative sample of the population and will be taken for the experiment as obtained by the breeding. If we observe single individual animals that obviously deviate from the population, where it can be unambiguously excluded that this deviation is caused by the mutation that is to be analyzed (e.g. hermaphrodites, long teeth as result from dysgnatia, or mice with injuries from fights with cage mates), and where the inclusion of this animal would systematically confound the experiment, are excluded as an a priori criterion.

Before the start of each individual experiment within the phenotyping pipeline every animal is examined for its physical condition. In case that the condition of the animal does not allow that the experiment can be performed in a safe and harmless manner for the animal, the test will be skipped for this animal. Additional exclusion criteria as well as stop criteria that are specific for each test are defined in the SOP for each test. For example, digit abnormalities exclude mice from grip. strength measurements. A test will be completely stopped as soon as two mice of the test cohort show any problems with the test conditions (e.g. anesthesia incompatibility in the X-ray test).

 

b. In general, no data point will be excluded from the analysis, unless there is clear evidence of e.g. technical failure of the experimental machine. For many of the parameters (like blood parameters) we have pre-established threshold values as indicators for invalid measurements. If the data point reaches the criteria, we have to expect that there was an undetectable failure, and the data point is replaced by a status code (e.g. above upper limit).

 

c.

Screen

Parameterset

number in each experimental group

Behaviour

Openfield

15

Neurology

SHIRPA, Grip strength, Rotarod

15

Behaviour

Acoustic startle / PPI

15

Clinical Chemistry

Clinical chemistry after fasting

15

Dysmorphology

Anatomical observation

15

Allergy

Transepidermal water loss (TEWL), Body surface temperature (BST) , Alopecia

15

Nociception

Hotplate

15

Metabolism

Body composition, Indirect calorimetry

15

Clinical Chemistry

Ip-Glucose-tolerance-test

15

Cardio

ECG / Echocardiography

15

Eye and Vision

OCT, Laser-interference-biometry

Scheimpflug, Optokinetic drum

15

7

Metabolism

Body composition

15

Bone and Cartilage

X-Ray, Bone densitometry (DXA)

15

Neurology

ABR (Auditory brain stem response)

10

Immunology

Flow Cytometry, Immunoglobulin isotyping

15

Allergy

Panel MSD (IgE, IL6, TNF, Insulin, Leptin, FGF-21)

15

Clinical Chemistry

Clinical chemistry, Haematology

15

Pathology

Histopathology, Gross pathology, Tissue embedding and block banking, Organ weights

5

Molecular Phenotyping

Expression Profiling

10

 

 

4. Randomisation

Recommendation

a. State whether randomization was used to allocate experimental units to control and treatment groups. If done, provide the method used to generate the randomization sequence.

b. Describe the strategy used to minimize potential confounders such as the order of treatments and measurements, or animal/cage location. If confounders were not controlled, state this explicitly.

GMC Approach

b) We control for all known influencing factors: our mice are maintained under specific pathogen-free conditions with standardized diet, acclimatization and standardized cage enrichment. All mice tested are located in the same rack within the housing room.

Female and male mice are separated in the cages, genotypes are mixed. Cage sizes are between 2 and 5 animals usually. The maximum age difference between youngest and oldest animal is 7 days.

To minimize potential confounders during the measurement, we standardize external factors as much as possible. All mice of a project will usually be measured concurrently with the same machine and by the same technician.

When it comes to testing the mice, we ensure that selection of the mice is balanced, meaning, for example, that we measure control and mutants in parallel or alternately. In tests where the order of the mice has an influence on the results (e.g. earlier versus later sample taking), we create lists defining the order of the measured mice that are balanced for the experimental and the control group.

Blood samples are randomized by the robot (the robot includes a specific programing for randomization of the samples in the plate).

 

 

5. Blinding

Recommendation

Describe who was aware of the group allocation at the different stages of the experiment (during the allocation, the conduct of the experiment, the outcome assessment, and the data analysis).

GMC Approach

In most tests, blinding is not needed, as the experimenter has no influence on the results of these tests, since the results are directly recorded by the machine. In the tests, where the experimenter might have an influence on the measurements, the experimenter is blinded, and we have SOP's prescribing how this will be ensured for each single test separately. Blood samples are blinded (the robot works with barcodes, not IDs of the mice), preparation by automation. In addition, we record metadata for each data point and monitor the influence of these metadata over time.

In some cases, blinding is not possible, e.g. when the genotype is obvious by simply looking at the mice.

Data analysis is automated via standardized R-scripts.

 

 

6. Outcome Measures

Recommendation

a. Clearly define all outcome measures assessed (e.g. cell death, molecular markers, or behavioural changes). b. For hypothesis-testing studies, specify the primary outcome measure, i.e. the outcome measure that was used to determine the sample size.

GMC Approach

a) We measure more than 500 parameters in all screens.

 

Screen

Parameterset

Number of parameters measured

Standard Method for statistical analysis

Behaviour

Openfield

39

2-way ANOVA

Neurology

 

 

 

Shirpa

21

Fishers' Exact Test, Linear Model

Grip strength

8

Linear Model

Rotarod

8

Linear Mixed Effects Model, Fishers' Exact Test

Behaviour

Accustic startle / PPI

19

2-way ANOVA

Clinical Chemistry

Clinical chemistry after fasting

6

Linear model for effects of genotype and sex and interaction of both,

2-way ANOVA with post-hoc Tukey test for effects of genotype and sex and interaction of both and genotype effects in each test group in case of interactions

Wilcoxon rank-sum test for separate analysis of genotype effects in males and females in case of skewed distribution of values.
Dysmorphology

Anatomical observation

70

Fishers Exact Test
AllergyTransepidermal water loss (TEWL)3Wilcoxon rank-sum test

Body surface temperature (BST) / Alopecia

3

Wilcoxon rank-sum test
NociceptionHotplate

7

2-way ANOVA, Fishers Exact Test
MetabolismBody composition

4

Linear Model
Indirect calorimetry

310

Linear Model

Clinical Chemistry

Ip-Glucose-tolerance-test

7

Linear model for effects of genotype and sex and interaction of both,

Wilcoxon rank-sum test for separate analysis of genotype effects in males and females

CardioECG

9

Wilcoxon rank-sum test
Echocardiography

5

Wilcoxon rank-sum test

Eye and vision

OCT

12

Wilcoxon rank-sum test,

Linear model for effects of genotype and sex and interaction of both

Laser-interference-biometry

10

Wilcoxon rank-sum test,

Linear model for effects of genotype and sex and interaction of both

Scheimpflug

8

Wilcoxon rank-sum test
Optokinetic drum

2

Wilcoxon rank-sum test
MetabolismBody composition

4

Linear Model
Bone and Cartilage

 

X-Ray

38

Fishers exact test
DXA

16

Fishers exact test
NeurologyABR (Auditory brain stem response)

8

Wilcoxon-rank-sum test, Linear Mixed Effects Model

Immunology

 

Flow Cytometry

50

Wilcoxon rank-sum test

Immunoglobulin isotyping

5

Wilcoxon rank-sum test
AllergyPanel MSD (IgE, IL6, TNF, Insulin, Leptin, FGF-21)

6

Wilcoxon rank-sum test
Clinical Chemistry

 

Clinical Chemistry

20-23

Linear model for effects of genotype and sex and interaction of both,

2-way ANOVA with post-hoc Tukey test for effects of genotype and sex and interaction of both and genotype effects in each test group in case of significant genotype x sex interactions

Wilcoxon rank-sum test for separate analysis of genotype effects in males and females in case of skewed distribution of values.

Haematology

4

Linear model for effects of genotype and sex and interaction of both,

2-way ANOVA with post-hoc Tukey test for effects of genotype and sex and interaction of both and genotype effects in each test group in case of interactions

Wilcoxon rank-sum test for separate analysis of genotype effects in males and females in case of skewed distribution of values.

Pathology

Histopathology

Descriptive pathology

124

Fishers Exact Test, and in few cases semi-quantitative histological scores

Gross pathology /

Descriptive Terms

31

Fishers Exact Test

Tissue embedding and block banking

33

Not applicable (block banked, not block banked)
Organ weights

5-10

Wilcoxon rank-sum test or Linear Model
Molecular phenotyping

Expression Profiling

55.401 Transcripts

DEseq2 (Bioconductor)

 

b) Not applicable: No hypothesis testing study (screening project)

 

7. Statistical Methods

Recommendation

a. Provide details of the statistical methods used for each analysis, including software used.

b. Describe any methods used to assess whether the data met the assumptions of the statistical approach, and what was done if the assumptions were not met.

GMC Approach

a) Data is analyzed using automated R-scripts (software version 3.4.4   (2018-03-15) -- "Someone to Lean On").

Since we have phenotyping data of control animals for almost 20 years, we know the distribution of every single parameter. Based on this knowledge, we selected the best statistical test. We have a customized analysis for each parameter including also complex models (e.g. LME's, body-weight as additional covariate).

For the exact statistical methods used in each screen, please refer to table 6a)

 

b) Please see above. Distributions of the parameters are regularly checked. We implemented different quality control plots like QQ plots, histograms, KS test, effect sizes etc. in our automated R-scripts used to analyze the data.

 

8. Experimental Animals

Recommendation

a. Provide species-appropriate details of the animals used, including species, strain and sub strain, sex, age or developmental stage, and, if relevant, weight.

b. Provide further relevant information on the provenance of animals, health/immune status, genetic modification status, genotype, and any previous procedures.

GMC Approach

a) The genetic background of the animals is project specific (at least 3 generations controlled breeding (e.g. backcrossing) after mutation generation). The used female and male mice usually start the phenotyping pipeline from the age of 9 weeks until 18 weeks. Wild-type animals have to be littermates of the mutant animals. The maximum age difference between youngest and oldest is 7 days.

 

b) We request a health report for the mouse line according to the FELASA guidelines before start of the project. The decision whether we can accept the mice in our facility is done by independent veterinarians. In case that the mice would not be allowed to enter our facility, they will not even be bred (3Rs principle). A second, more recent, health report is requested before sending the mice.

The collaboration partner generates (kind of mutation is project specific) and breeds the cohort for the phenotypic analysis and provides the requested animal number (15 males and 15 females (mutants) and 15 males and 15 females (wild type)). The animals have to be sent when they are 7 weeks old, with complete genotype information.

 

9. Experimental Procedures

Recommendation

For each experimental group, including controls, describe the procedures in enough detail to allow others to replicate them, including:

a. What was done, how it was done and what was used.

b. When and how often.

c. Where (including detail of any acclimatization periods).

d. Why (provide rationale for procedures).

GMC Approach

a)  Within the screening pipeline (Gailus-Durner et al., 2005; Fuchs et al., 2009, Gailus-Durner et al., 2009, Fuchs et al., 2011, Fuchs et al., 2018) all clinically relevant organ-systems of the mice are analyzed systematically covering a broad range of tests to identify novel phenotypes. Procedures of the tests are indicated in each test-SOP (including information about e.g. calibration or quality control steps). Methods of the standardized primary pipeline are published (see table below).

 

Age (weeks)

Screen

TestReference for the protocol

9

Behaviour

Open Field test

Hölter SM et al (2015); PMID: 26629773

Garrett L et al (2012);  PMID: 22682077

9

Neurology

Modified SHIRPA, grip strengthFuchs et al., 2011; PMID: 20708688

10

Behaviour

Acoustic startle response and pre-pulse inhibitionHeermann et al. (2019); PMID: 30291584

10

NeurologyRotarod

Fuchs et al., 2011; PMID: 20708688

André at al., 2018; PMID: 29659570

11

Clinical Chemistry

Fasting plasma lipid and glucose values

Rathkolb B et al., 2013: PMID: 2606905922682077

12

Nociception

Hot plate test

Fuchs et al., 2011; PMID: 20708688

André at al., 2018; PMID: 29659570

Salminen et al., 2017; PMID: 28645892

12

Bone and CartilageMorphological observationFuchs et al., 2011; PMID: 20708688

12

AllergyTEWL / BST

Nicolaus et al., 2016; PMID:27657706

Rekant et al., 2016; PMID: 26709943

Fuchs et al., 2017; PMID: 28966146

13

MetabolismIndirect calorimetry and Body compositionFuchs et al., 2011; PMID: 20708688

14

Clinical ChemistryIntraperitoneal Glucose tolerance testRozman et al., 2015; PMID: 25727201

15

CardioAwake electro-cardiogram and echocardio-graphyMoreth et al., 2014; PMID: 24788387.

16

Eye and vision

Scheimpflug imaging, OCT, Laser-interference-biometry, Optokinetic drum

Puk et al., (2009) PMID: 19578028;

Pawliczek et al., (2019); PMID: 31751552

Puk et al., (2006); PMID: 16897341

Puk et al., (2008); PMID: 18508659

Benkner et al., (2013); PMID: 23957722

17

NeurologyAuditory brainstem response

Fuchs et al., 2011; PMID: 20708688

André at al., 2018; PMID: 29659570

Salminen et al., 2017; PMID: 28645892

Lee et al., 2015; PMID: 26464487

17

Bone and Cartilage

X-ray analysis,

Bone densitometry (DXA)

Fuchs et al., 2011; PMID: 20708688

Swan et al., 2020; PMID: 33370286

Ignatova et al., 2020; PMID: 32217665

18

MetabolismBody compositionFuchs et al., 2011; PMID: 20708688

19

Clinical chemistry

Clinical chemistry (ad lib. fed)Rathkolb B et al., 2013: PMID: 26069059

19

Clinical chemistryHematologyRathkolb B et al., 2013: PMID: 26069060

19

Immunology / Allergy

Flow Cytometry analysis of peripheral blood cells; MSD- immunoglobulins;

MSD-Biomarker-Panel

Fuchs et al., 2011; PMID: 20708688;

Daubeuf et al. 2017; PMID: 28628216

Aguilar-Pimentel et al., 2020; PMID: 33110090

19

PathologyMacro- and microscopic analysis

Fuchs et al., 2011; PMID: 20708688

Serpi, R et al., 2013; PMID: 23233061

Sibylle Sabrautzki et al., 2013; PMID: 2379184

 

b) The pipeline usually starts with animals at 9 weeks of age. Tests, order of the tests and frequency is depicted in the table above and in the workflow of the screening pipeline (https://www.mouseclinic.de/research/gmc-pipelines/screening-pipeline/index.html)

c) The animals are sent by the collaboration partner who performed the breeding and are directly transferred to the dedicated housing room in our SPF facility. The mice have an acclimatization period of 2 weeks before the first measurement. We have a dedicated lab room for each test procedure. Mice are housed in a housing room close to the lab room, and are brought to the lab for the test. Test-specific acclimatization periods are defined in SOPs.

d) not applicable

 

10. Results

Recommendation

For each experiment conducted, including independent replications, report:

a. Summary/descriptive statistics for each experimental group, with a measure of variability where applicable (e.g. mean and SD, or median and range).

b. If applicable, the effect size with a confidence interval.

GMC Approach

a/b) For each parameter, we calculate measures like mean, standard-deviation, effect size and respectively median and IQR in our standardized automatic R-scripts.

 

 

 

The Recommended Set

These items complement the Essential 10 and add important context to the study. Reporting the items in both sets represents best practice.

 

Ethical Statement

Recommendation

Provide the name of the ethical review committee or equivalent that has approved the use of animals in this study, and any relevant license or protocol numbers (if applicable). If ethical approval was not sought or granted, provide a justification.

GMC Approach

The GMC holds a general license to run phenotype assessments in mice and all tests are approved by the responsible authority of the district government of Upper Bavaria. Individual projects are registered and approved by the government approximately 4 weeks before start.

 

Housing and Husbandry

Recommendation

Provide details of housing and husbandry conditions, including any environmental enrichment.

GMC Approach

Animal housing is performed in strict accordance with directive 2010/63/EU and the local government. All mice are housed in individually ventilated caging (IVC) systems (Sealsafe plus, GM 500, Tecniplast, Buggugiate, Italy) under specific pathogen-free conditions (GMC) with a maximum cage density of five adult mice per cage. IVC systems operate with positive pressure. All mice receive autoclaved wood chips (Lignocel select fine, J. Rettenmaier & Soehne GmbH, Rosenberg, Germany) and paper stripes (Arbocel crinclets natural Rettenmaier & Soehne GmbH) as bedding and nesting material, enrichment (red houses),irradiated standard diet for rodents (Altromin 1314, Altromin Spezialfutter GmbH, Lage, Germany) and sterile-filtered tap water ad libitum. Mice are transferred to new cages in laminar flow cage changing stations in weekly intervals. Light is adjusted to a 12h/12h light/dark cycle with a 10 min period of dimmed light to simulate sunrise/sunset; temperature and relative humidity are regulated to 22 ± 2° C and 55 ± 10 %. Access via air sluices is restricted by an electronic key system, and it is mandatory to wear personal protective equipment (autoclaved trousers and shirts, dedicated shoes, surgical masks, hair bonnets and gloves). Health monitoring is based on quarterly exhaust air dust PCR analysis for all FELASA-listed agents by a commercial diagnostic laboratory (Mähler M et al., 2014). All mice tested are located in the same room and rack.

Data Access

Recommendation

Provide a statement describing if and where study data are available.

GMC Approach

Raw data can be requested (the data will be transmitted via fixed data formats such as CSV/XML).

Upload data of single knockout lines to the IMPC webpage ( https://www.mousephenotype.org )- the data can be downloaded directly from the webpage (https://www.mousephenotype.org/understand/accessing-the-data/). 

The phenotyping results of the analyzed mouse lines can be uploaded to our Open Access GMC phenomap (https://www.mouseclinic.de/– see results).

Upload the data to a generic repository on demand.