Essential 10

8. Experimental animals

The species, strain, substrain, sex, weight, and age of animals are critical factors that can influence most experimental results [1-5]. Reporting the characteristics of all animals used is equivalent to standardised human patient demographic data; these data support both the internal and external validity of the study results. It enables other researchers to repeat the experiment and generalise the findings. It also enables readers to assess whether the animal characteristics chosen for the experiment are relevant to the research objectives.

When reporting age and weight, include summary statistics for each experimental group (e.g. mean and standard deviation) and, if possible, baseline values for individual animals (e.g. as supplementary information or a link to a publicly accessible data repository). As body weight might vary during the course of the study, indicate when the measurements were taken. For most species, precise reporting of age is more informative than a description of the developmental status (e.g. a mouse referred to as an adult can vary in age from six to twenty weeks [6]). In some cases, however, reporting the developmental stage is more informative than chronological age, for example in juvenile Xenopus, where rate of development can be manipulated by incubation temperature [7].

Reporting the weight or the sex of the animals used may not feasible for all studies. For example, sex may be unknown for embryos or juveniles, or weight measurement may be particularly stressful for some aquatic species. If reporting these characteristics can be reasonably expected for the species used and the experimental setting but are not reported, provide a justification. 

 

References

  1. Clayton JA and Collins FS (2014). Policy: NIH to balance sex in cell and animal studies. Nature News. doi: 10.1038/509282a
  2. Shapira S, Sapir M, Wengier A, Grauer E and Kadar T (2002). Aging has a complex effect on a rat model of ischemic stroke. Brain Res. doi: 10.1016/s0006-8993(01)03270-x
  3. Vital M, Harkema JR, Rizzo M, Tiedje J and Brandenberger C (2015). Alterations of the murine gut microbiome with age and allergic airway disease. J Immunol Res. doi: 10.1155/2015/892568
  4. Bouwknecht JA and Paylor R (2002). Behavioral and physiological mouse assays for anxiety: a survey in nine mouse strains. Behavioural brain research. doi: 10.1016/s0166-4328(02)00200-0
  5. Simon MM, Greenaway S, White JK, Fuchs H, Gailus-Durner V, Wells S, Sorg T, Wong K, Bedu E, Cartwright EJ, Dacquin R, Djebali S, Estabel J, Graw J, Ingham NJ, Jackson IJ, Lengeling A, Mandillo S, Marvel J, Meziane H, Preitner F, Puk O, Roux M, Adams DJ, Atkins S, Ayadi A, Becker L, Blake A, Brooker D, Cater H, et al. (2013). A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains. Genome Biol. doi: 10.1186/gb-2013-14-7-r82
  6. Jackson SJ, Andrews N, Ball D, Bellantuono I, Gray J, Hachoumi L, Holmes A, Latcham J, Petrie A, Potter P, Rice A, Ritchie A, Stewart M, Strepka C, Yeoman M and Chapman K (2017). Does age matter? The impact of rodent age on study outcomes. Laboratory Animals. doi: 10.1177/0023677216653984
  7. Khokha MK, Chung C, Bustamante EL, Gaw LW, Trott KA, Yeh J, Lim N, Lin JC, Taverner N, Amaya E, Papalopulu N, Smith JC, Zorn AM, Harland RM and Grammer TC (2002). Techniques and probes for the study of Xenopus tropicalis development. Dev Dyn. doi: 10.1002/dvdy.1018 

Example 1 

“One hundred and nineteen male mice were used: C57BL/6OlaHsd mice (n = 59), and BALB/c OlaHsd mice (n = 60) (both from Harlan, Horst, The Netherlands). At the time of the EPM test the mice were 13 weeks old and had body weights of 27.4 ± 0.4 g and 27.8 ± 0.3 g, respectively (mean ± SEM).” [1]

Example 2 

“Histone Methylation Profiles and the Transcriptome of X. tropicalis Gastrula Embryos. To generate epigenetic profiles, ChIP was performed using specific antibodies against trimethylated H3K4 and H3K27 in Xenopus gastrula-stage embryos (Nieuwkoop-Faber stage 11–12), followed by deep sequencing (ChIP-seq). In addition, polyA-selected RNA (stages 10–13) was reverse transcribed and sequenced (RNA-seq).” [2] 

 

References

  1. Okva K, Nevalainen T and Pokk P (2013). The effect of cage shelf on the behaviour of male C57BL/6 and BALB/c mice in the elevated plus maze test. Lab Anim. doi: 10.1177/0023677213489280
  2. Akkers RC, van Heeringen SJ, Jacobi UG, Janssen-Megens EM, Francoijs KJ, Stunnenberg HG and Veenstra GJ (2009). A hierarchy of H3K4me3 and H3K27me3 acquisition in spatial gene regulation in Xenopus embryos. Dev Cell. doi: 10.1016/j.devcel.2009.08.005

The animals’ provenance, their health or immune status and their history of previous testing or procedures, can influence their physiology and behaviour as well as their response to treatments, and thus impact on study outcomes. For example, animals of the same strain, but from different sources, or animals obtained from the same source but at different times, may be genetically different [1]. The immune or microbiological status of the animals can also influence welfare, experimental variability and scientific outcomes [2-4].

Report the health status of all animals used in the study, and any previous procedures the animals have undergone. For example, if animals are specific pathogen free (SPF), list the pathogens that they were declared free of. If health status is unknown or was not tested, explicitly state this.

For genetically modified animals, describe the genetic modification status (e.g. knockout, overexpression), genotype (e.g. homozygous, heterozygous), manipulated gene(s), genetic methods and technologies used to generate the animals, how the genetic modification was confirmed, and details of animals used as controls (e.g. littermate controls [5]).

Reporting the correct nomenclature is crucial to understanding the data and ensuring that the research is discoverable and replicable [6-8]. Useful resources for reporting nomenclature for different species include:

 

References

  1. Festing MF and Altman DG (2002). Guidelines for the design and statistical analysis of experiments using laboratory animals. ILAR journal. http://www.ncbi.nlm.nih.gov/pubmed/12391400
  2. Mahler Convenor M, Berard M, Feinstein R, Gallagher A, Illgen-Wilcke B, Pritchett-Corning K and Raspa M (2014). FELASA recommendations for the health monitoring of mouse, rat, hamster, guinea pig and rabbit colonies in breeding and experimental units. Lab Anim. doi: 10.1177/0023677213516312
  3. Baker DG (1998). Natural Pathogens of Laboratory Mice, Rats, and Rabbits and Their Effects on Research. Clinical Microbiology Reviews. doi: 10.1128/cmr.11.2.231
  4. Velazquez EM, Nguyen H, Heasley KT, Saechao CH, Gil LM, Rogers AWL, Miller BM, Rolston MR, Lopez CA, Litvak Y, Liou MJ, Faber F, Bronner DN, Tiffany CR, Byndloss MX, Byndloss AJ and Baumler AJ (2019). Endogenous Enterobacteriaceae underlie variation in susceptibility to Salmonella infection. Nat Microbiol. doi: 10.1038/s41564-019-0407-8
  5. Holmdahl R and Malissen B (2012). The need for littermate controls. European journal of immunology. doi: 10.1002/eji.201142048
  6. Mallapaty S (2018). In the name of reproducibility. Lab Animal. doi: 10.1038/s41684-018-0095-7
  7. Sundberg JP and Schofield PN (2010). Commentary: Mouse Genetic Nomenclature:Standardization of Strain, Gene, and Protein Symbols. Veterinary Pathology. doi: 10.1177/0300985810374837
  8. Montoliu L and Whitelaw CBA (2011). Using standard nomenclature to adequately name transgenes, knockout gene alleles and any mutation associated to a genetically modified mouse strain. Transgenic Research. doi: 10.1007/s11248-010-9428-z

Example 1

“A construct was engineered for knock-in of the miR-128 (miR-128-3p) gene into the Rosa26 locus. Rosa26 genomic DNA fragments (~1.1 kb and ~4.3 kb 5′ and 3′ homology arms, respectively) were amplified from C57BL/6 BAC DNA, cloned into the pBasicLNeoL vector sequentially by in-fusion cloning, and confirmed by sequencing. The miR-128 gene, under the control of tetO-minimum promoter, was also cloned into the vector between the two homology arms. In addition, the targeting construct also contained a loxP sites flanking the neomycin resistance gene cassette for positive selection and a diphtheria toxin A (DTA) cassette for negative selection. The construct was linearized with ClaI and electroporated into C57BL/6N ES cells. After G418 selection, seven-positive clones were identified from 121 G418-resistant clones by PCR screening. Six-positive clones were expanded and further analyzed by Southern blot analysis, among which four clones were confirmed with correct targeting with single-copy integration. Correctly targeted ES cell clones were injected into blastocysts, and the blastocysts were implanted into pseudo-pregnant mice to generate chimeras by Cyagen Biosciences Inc. Chimeric males were bred with Cre deleted mice from Jackson Laboratories to generate neomycin-free knockin mice. The correct insertion of the miR-128 cassette and successful removal of the neomycin cassette were confirmed by PCR analysis with the primers listed in Supplementary Table 1.” [1]

Example 2

“The C57BL/6J (Jackson) mice were supplied by Charles River Laboratories. The C57BL/6JOlaHsd (Harlan) mice were supplied by Harlan. The α-synuclein knockout mice were kindly supplied by Prof. [X] (Cardiff University, Cardiff, United Kingdom.) and were congenic C57BL/6JCrl (backcrossed for 12 generations). TNFα−/− mice were kindly supplied by Dr. [Y] (Queens University, Belfast, Northern Ireland) and were inbred on a homozygous C57BL/6J strain originally sourced from Bantin & Kingman and generated by targeting C57BL/6 ES cells. T286A mice were obtained from Prof. [Z] (University of California, Los Angeles, CA). These mice were originally congenic C57BL/6J (backcrossed for five generations) and were then inbred (cousin matings) over 14 y, during which time they were outbred with C57BL/6JOlaHsd mice on three separate occasions.” [2]

 

References

  1. Huang W, Feng Y, Liang J, Yu H, Wang C, Wang B, Wang M, Jiang L, Meng W, Cai W, Medvedovic M, Chen J, Paul C, Davidson WS, Sadayappan S, Stambrook PJ, Yu XY and Wang Y (2018). Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration. Nature communications. doi: 10.1038/s41467-018-03019-z
  2. Ranson A, Cheetham CEJ, Fox K and Sengpiel F (2012). Homeostatic plasticity mechanisms are required for juvenile, but not adult, ocular dominance plasticity. Proceedings of the National Academy of Sciences. doi: 10.1073/pnas.1112204109