The present review aims to summarize the main features of mammary gland anatomy, and the physiology of lactation and colostrum/milk in the most commonly used animal species for regulatory toxicity. The final goal is the selection of a preferred animal species to be enrolled in studies investigating the potential transfer of drugs and exogenous molecules through milk, within the Innovative Medicines Initiative (IMI) funded project ConcePTION. Reference data regarding humans were also collected and analyzed in order to highlight critical similarities and differences with the studied species. Additional practical considerations were also taken into account, such as ethical consideration regarding the chosen species which affects the group size, financial implications and technical feasibility of lactation trials (e.g., ease of sampling, volume of sampling, husbandry requirements and scientific recognition). In conclusion, the present analysis of the literature confirms the complexity of the decisional process behind the choice of an animal model for in vivo trials. For some of the evaluated species, data were either poor or missing, highlighting the necessity to generate more physiological background studies for species that are routinely used in laboratory settings. Overall, when taking into consideration ethical factors, feasible group size, milk volume and ease of milk collection, and physiological similarities with humans, minipigs seem to represent the most appropriate choice.

Steroids, providing information regarding several biological patterns including stress and sexual behavior, have been investigated in different matrices in laboratory mice. Data regarding hair quantification, indicative of longer timespans when compared to blood and saliva, are lacking. The aim of the work was to analyze the hormonal hair profile of laboratory male mice and to investigate potential relationships with age and housing, as a potential tool for welfare assessment. Fifty-six adult male C57BL/6J and C57BL/6OlaHsd substrain mice were included in the study, housed in pairs or groups. Testosterone (T) and dehydroepiandrosterone (DHEA) were quantified by radioimmunoassay, corticosterone (CORT) by ELISA. Mean hormone levels were 6.42 pg/mg for T, 23.16 pg/mg for DHEA and 502.1 pg/mg for CORT. Age influenced all hormones by significantly increasing T and DHEA levels and decreasing CORT; only DHEA, significantly higher in grouped mice, was influenced by housing conditions. The influence of age indicates the need for accurate age-related reference intervals, while the higher levels of DHEA in grouped animals suggests that such housing practice may be beneficial for social interactions. In conclusion, it seems that hair hormones quantification may be a good tool for welfare assessment in laboratory mice and may help in refining husbandry.

The red deer (Cervus elaphus L., 1758) is one of the largest deer species in the world. Females are seasonal polyestrous, with negative photoperiod: the increase of the night peak of melatonin determines the secretion of GnRH and, therefore, LH and FSH. To date there is little information regarding the hormonal control during pregnancy for this species; this could be due to the difficulty of sampling wild subjects, while farmed animals’ hormonal concentrations may not reflect the physiology of the animal in a natural state. In this study we evaluated the concentration of cortisol and progesterone, extracted from blood and hair, on 10 wild and pregnant red deer females. Belonging to the population of the Bolognese Apennines (Italy), the hinds were sampled in the January–March 2018 period, according to the regional selective hunting plan. Plasma progesterone (P4) ranged from a minimum of 1.9 to a maximum of 7.48 ng/mL; while hair P4 concentrations varied from 41.68 to 153.57 pg/mg. The plasma and hair cortisol ranges are respectively 0.4–2.97 ng/mL and 0.03–0.55 pg/mg; the only significant correlation was found between hair concentration of P4 and the date of death. The results of this preliminary study represent a small step towards a better knowledge of this species’ physiology during pregnancy.

Background The use of adrenocorticotropic hormone stimulation test as method to monitor efficacy of trilostane treatment of hypercortisolism (HC) in dogs has been questioned. Objectives To evaluate and compare 12 methods with which to monitor efficacy of trilostane treatment in dogs with HC. Animals Forty‐five client‐owned dogs with HC treated with trilostane q12h. Methods Prospective cross‐sectional observational study. The dogs were categorized as well‐controlled, undercontrolled, and unwell through a clinical score obtained from an owner questionnaire. The ability to correctly identify trilostane‐treatment control of dogs with HC with the following variables was evaluated: before trilostane serum cortisol (prepill), before‐ACTH serum cortisol, post‐ACTH serum cortisol, plasma endogenous ACTH concentrations, prepill/eACTH ratio, serum haptoglobin (Hp) concentration, serum alanine aminotransferase (ALT), gamma‐glutamyl transferase (γGT) and alkaline phosphatase activity, urine specific gravity, and urinary cortisol : creatinine ratio. Results Ninety‐four re‐evaluations of 44 dogs were included; 5 re‐evaluations of 5 unwell dogs were excluded. Haptoglobin was significantly associated with the clinical score (P < .001) and in the receiver operating characteristic analysis, Hp cutoff of 151 mg/dL correctly identified 90.0% of well‐controlled dogs (specificity) and 65.6% of undercontrolled dogs (sensitivity). Alanine aminotransferase (P = .01) and γGT (P = .009) were significantly higher in undercontrolled dogs. Cutoff of ALT and γGT greater than or equal to 86 U/L and 5.8 U/L, respectively, were significantly associated with poor control of HC by trilostane. Conclusions and Clinical Importance Of all the 12 variables, Hp, and to a lesser degree ALT and γGT, could be considered additional tools to the clinical picture to identify well‐controlled and undercontrolled trilostane‐treated dogs.

The first weeks of life represent a crucial stage for microbial colonization of the piglets’ gastrointestinal tract. Newborns’ microbiota is unstable and easily subject to changes under stimuli or insults. Nonetheless, the administration of antibiotics to the sow is still considered as common practice in intensive farming for pathological conditions in the postpartum. Therefore, transfer of antibiotic residues through milk may occurs, affecting the piglets’ colon microbiota. In this study, we aimed to extend the knowledge on antibiotic transfer through milk, employing an in vitro dedicated piglet colon model (MICODE—Multi Unit In vitro Colon Model). The authors’ focus was set on the shifts of the piglets’ microbiota composition microbiomics (16S r-DNA MiSeq and qPCR—quantitative polymerase chain reaction) and on the production of microbial metabolites (SPME GC/MS—solid phase micro-extraction gas chromatography/mass spectrometry) in response to milk with different concentrations of amoxicillin. The results showed an effective influence of amoxicillin in piglets’ microbiota and metabolites production; however, without altering the overall biodiversity. The scenario is that of a limitation of pathogens and opportunistic taxa, e.g., Staphylococcaceae and Enterobacteriaceae, but also a limitation of commensal dominant Lactobacillaceae, a reduction in commensal Ruminococcaceae and a depletion in beneficial Bifidobactericeae. Lastly, an incremental growth of resistant species, such as Enterococcaceae or Clostridiaceae, was observed. To the authors’ knowledge, this study is the first evaluating the impact of antibiotic residues towards the piglets’ colon microbiota in an in vitro model, opening the way to include such approach in a pipeline of experiments where a reduced number of animals for testing is employed. Key points • Piglet colon model to study antibiotic transfer through milk. • MICODE resulted a robust and versatile in vitro gut model. • Towards the “3Rs” Principles to replace, reduce and refine the use of animals used for scientific purposes (Directive 2010/63/UE). Graphical abstract