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Gastrointestinal Microbiota & Reproducibility of Animal Disease Models

Reproducibility of preclinical research has been widely recognized as an important issue. The National Institutes of Health (NIH) launched several initiatives in 2014 to improve reproducibility (1), focusing on educating researchers and journals about appropriate experimental design and reporting of data to increase the transparency of research findings in published manuscripts. While these initiatives have certainly resulted in better planning and reporting of experiments, there are still research areas where reproducibility is an issue, namely, preclinical animal model research.

There are many sources of variation in animal model research, from the manufacturer of reagents and experimental animal strain selection, to subtle changes in protocol that can affect the disease phenotype and influence research findings. With the rise of 16S rRNA sequencing, gastrointestinal microbes of experimental animals have been implicated in phenotypic variance of disease models. For instance, in a series of intricate experiments, Ivanov and Wu et al. showed that specific microbiota (notably segmented filamentous bacteria or SFB) can induce differentiation of Th17 cells in the small intestine of mice (2,3) and that SFB can drive autoimmune arthritis models (4). Preclinical research of other inflammatory diseases has shown that microbiota modulates disease phenotype in experimental animal models of SLE (5,6), allergic asthma (7,8), gastrointestinal and metabolic disorders (9,10), and Sjögren Syndrome (11).

Sources of Microbial Variation

Given this influence, many researchers argue that variations in intestinal microbiota composition contribute to the low reproducibility of animal model studies (12-14). Even using mice of the same strain as a previously published study to minimize microbial variation is problematic, as vendor effects on microbiota composition are significant (8, 15-17). Differences between facilities within the same animal vendor can prove to have a substantial impact on the microbial profile as well (15,18,19). Further complicating matters is the influence of changes in diet (13,14), treatment and type of caging, bedding, and food/water (17,20), barrier access (17), and stress from recent transportation (13,14,21) on the microbiota composition.

With so many factors that can affect the intestinal microbial profile in experimental animals, and therefore experimental disease phenotype, it is no wonder that reproducing animal model research is notoriously difficult. Improvements in preclinical animal model research have been proposed to address this issue: longer acclimation periods to ensure a stable intestinal microbiota (21), increasing heterogeneity of studies by incorporating multi-laboratory study designs (22), and increasing study sizes to include a sufficient number of individuals from various animal vendors (13). While these steps may save money over time by increasing reproducibility and the applicability of experimental results, they also would drastically increase the operating costs of preclinical studies.

To improve the reproducibility of your experiments and to prevent any unforeseen changes in disease phenotype caused by compositional changes in intestinal microbiota, Chondrex, Inc. can provide a few recommendations.

Ways to Minimize Variations in Microbiota

1.    Use SPF housing conditions whenever possible.
Using SPF housing conditions can limit environmental exposure of pathogens and is highly recommended to avoid microbiota-dependent changes in disease phenotype. If SPF conditions are not possible at your facility, we recommend cleaning your animal facility thoroughly before mice arrive and throughout the duration of your experiment. 

2.    Perform a small trial study prior to a large-scale study.
Chondrex, Inc. always recommends running a small pilot study to evaluate animals from different vendors, prior to a full-scale experiment, to determine the disease susceptibility of animals from each vendor. Once you find an animal vendor and strain that works for your study, stick with it. If you can, request future animals come from the same facility as in your trial studies. In our own studies, we have noted differences in disease susceptibility based on the specific facility an animal originates from.

3.    Evaluate the microbial profile of experimental animals. 
Monitoring the microbial exposure of experimental animals, both before entering your facility and during your studies, can be a useful tool to understanding any variations in disease phenotype. In instances where 16S rRNA sequencing is not available, Chondrex, Inc.’s Mouse Anti-Bacterial Antibody Assay Kits and Bacterial Toxin Detection Assay Kits can be used to evaluate immune response to common commensal bacteria and their toxins.

Chondrex, Inc. would like to help you overcome the difficulties of preclinical animal model research. Our expert team of scientists are always available to assist you with establishing your experimental protocol or troubleshooting any problems you may encounter. If you ever have any questions or concerns regarding your preclinical animal model research, please do not hesitate to contact us.      


  1. F. Collins, L. Tabak, Policy: NIH plans to enhance reproducibility. Nature 505, 612-613 (2014).
  2. I. Ivanov et al., Specific microbiota direct the differentiation of IL-17 producing T-helper cells in the mucosa of the small intestine. 4, 334-349 (2008).
  3. I. Ivanov et al., Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485-498 (2010).
  4. H.-J. Wu et al., Gut-Residing Segmented Filamentous Bacteria Drive Autoimmune Arthritis via T Helper 17 Cells. Immunity 32, 815-827 (2010).
  5. Y. Ma et al., Gut microbiota promote the inflammatory response in the pathogenesis of systemic lupus erythematosus. Molecular Medicine 25, (2019).
  6. S.-C. Choi, M. Mohmadzadeh, L. Morel, Gut dysbiosis contributes to autoimmune pathogenesis in lupus-prone mice. J Immunol 198, 58.52 (2017).
  7. M. Sokolowska, R. Frei, N. Lunjani, C. Akdis, L. O'Mahony, Microbiome and asthma. Asthma Res Pract 4, (2018).
  8. H.-Y. S. Chang, W. Mitzner, J. Watson, Variation in the airway responsiveness of male C57BL/6 mice from 5 vendors. Journal of the American Association for Laboratory Animal Science 51, 401-406 (2012).
  9. C. Chang, H. Lin, Dysbiosis in gastrointestinal disorders. Best Practice & Research Clinical Gastroenterology 30, 3-15 (2016).
  10. K. Brown, D. DeCoffe, E. Molcan, D. Gibson, Diet-induced dysbiosis of the intestinal microbiota and the effects on immunity and disease. Nutrients 4, 1095-1119 (2012).
  11. C. de Pavia et al., Altered mucosal microbiome diversity in disease severity in Sjögren syndrome. Scientific Reports 6, 23561 (2016).
  12. M.-L. Alegre, Mouse microbiomes: overlooked culprits of experimental variability. Genome Biology 20, 108 (2019).
  13. P. Turner, The role of gut microbiota on animal model reproducibility. Animal Model Exp Med 1, 109-115 (2018).
  14. C. Franklin, A. Ericsson, Microbiota and reproducibility of rodent models. Lab Anim (NY) 46, 114-122 (2017).
  15. K. Parker, S. Albeke, J. Gigley, A. Goldstein, N. Ward, Microbiome composition in both wild-type and disease model mice is heavily influenced by mouse facility. Front Microbiol 9, 1598 (2018).
  16. A. Ericsson et al., Effects of vendor and genetic background on the composition of the fecal microbiota of inbred mice. PLoS ONE 10, (2015).
  17. M. Hufeldt, D. Nielsen, F. Vogensen, T. Midtvedt, A. Hansen, Variation in the gut microbiota of laboratory mice is related to both genetic and environmental factors. Comparative Medicine 60, (2010).
  18. P. Rausch et al., Analysis of factors contributing to variation in the C57BL/6 fecal microbiota across german animal facilities. International Journal of Medical Microbiology 306, 343-355 (2016).
  19. G. Rogers et al., Functional divergence in the gastrointestinal microbiota in physically-separated genetically identical mice. Scientific Reports 4, 5437 (2014).
  20. A. Ericsson et al., The influence of caging, bedding, and diet on the composition of the microbiota in different regions of the mouse gut. Scientific Reports 8, 4065 (2018).
  21. D. Montonye et al., Acclimation and institutionalization of the mouse microbiota folloowing transportation. Front Microbiol 9, 1085 (2018).
  22. B. Voelkl, L. Vogt, E. Sena, H. Würbel, Reproducibility of preclinical animal research improves with heterogeneity of study samples. PLoS Biol 16, e2003693 (2018).


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