Fuochi, Sara (2022) Phenotypic characterization of wild-type animals to bridge a crucial knowledge gap. [Tesi di dottorato]

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An accurate understanding of genetic and phenotypic traits of laboratory animal models is crucial to securing validity of scientific results and animal welfare. The phenotyping pipeline is well established and proactively performed by research institutions, repository and commericial suppliers worldwide most typically on genetically modified animal models, also in the light of regulatory requirements. Despite the same pipeline is applicable as well to non genetically modified models, the level of in-depth phenotypical characterisation of stock, non mutant rodents strain, is not as systematic as for mutant rodents. In an effort to widening the characterisation of non-GM rodents phenotype, we focused on two aspects that were either non-characterised or only partially characterised in the pioneering phase of the animal strain. Particularly, with reference to the laboratory mouse, we undertook a study to disentangle the diurnal activity and feature key aspects of three non-genetically altered mouse strains widely used in research, C57BL/6NCrl (inbred), BALB/cAnNCrl (inbred) and CRL:CD1(ICR) (outbred). With this aim, we conducted a longitudinal analysis of the spontaneous locomotor activity of the mice during a 24-h period for 2 months, in two different periods of the year to reduce the seasonality effect. Mice (males and females) were group-housed in Digital Ventilated Cages (Tecniplast), mimicking standard housing conditions in research settings and avoiding the potential bias provided in terms of locomotor activity by single housing. The recorded locomotor activity was analyzed by relying on different and commonly used circadian metrics (i.e., day and night activity, diurnal activity, responses to lights-on and lights-off phases, acrophase and activity onset and regularity disruption index) to capture key behavioral responses for each strain. Our results clearly demonstrate significant differences in the circadian activity of the three selected strains, when comparing inbred versus outbred as well as inbred strains (C57BL/6NCrl versus BALB/cAnNCrl). Conversely, males and females of the same strain displayed similar motor phenotypes; significant differences were recorded only for C57BL/6NCrl and CRL:CD1(ICR) females, which displayed higher average locomotor activity from prepuberty to adulthood. All strain-specific differences were further confirmed by an unsupervised machine learning approach. Altogether, our data corroborate the concept that each strain behaves under characteristic patterns, which needs to be taken into consideration in every study design to ensure experimental reproducibility and comply with essential animal welfare principles. Furthermore, with reference to the laboratory rat, and considering the relevance of exact timing of puberty in preclinical studies, we developed a novel males puberty onset curve, performing a population screening of two outbred strains. Extensive bibliographic resources highlight that in male rats, the age of sexual maturity varies considerably between 40 and 60 days of age. Our screening allowed us to perform a thorough pubertal onset evaluation of Crl:CD(SD) and Crl:LE, relying on the balano-preputial separation test (BPS). Evaluation was carried out on animals under standard barrier conditions, from 4 to 9 weeks of age. In the Crl:CD(SD) population, 90% of males gained the puberty at week 6, and 100% in the following weeks whereas 75% of Crl:LE reached the puberty at week 6, 90% at week 7 and 100% from week 8. Remarkably, in both strains, puberty onset was gained at the average weight of 200 gr suggesting that weight range, not only age range, can be considered a biomarker of puberty onset in these two strains. On the contrary, descended testes cannot be considered an additional factor to identify the full puberty onset either in Crl:CD(SD) and Crl:LE rats. As a whole, the works reported in this thesis contributed to a better understanding of stock, non genetically modified models. Both studies were succesfully published, confirming the interest of the in vivo research community for the phenotypical assessment of commercially available non mutant rodents strains.

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