Hair steroid quantification: a non-invasive method to asses reproductive and welfare in wild and laboratory species .

The Hypothalamic-Pituitary-Gonadal (HPG) and Hypothalamic-Pituitary-Adrenal (HPA) axis are the main pathways responsible for behavioural modulation and reproduction[1]. In animal studies, steroids have mainly been analysed and quantified on several matrices. However, hair steroids have been studied and analysed in numerous species including domestic animal, carnivorous, rodents, livestock and cervids in wild and laboratory setting [2], [3], [4], [5]. Especially for studies in wildlife and lab species, animal who are difficult to hold or difficult to sampling high quantity [6]. Hair from 4 different species were collected: 28 sexually mature roe deer bucks (Capreolus capreolus), 10 pregnant female red deer (Cervus elaphus), 20 deceased (11 male and 9 female, young, sub-adult and adult) Appenine wolf (Canis lupus italicus) and 56 sexually mature male mice (Mus musculus). Briefly, hair was washed with water and propanol-2-ol. Once fully dried, hair was finely pulverized. Each pulverized sample was incubated overnight with methanol for steroids extraction. After centrifugation, methanol was collected and evaporated to dryness under an air-stream suction hood. All steroids quantification was performed with radioimmunoassy (RIA) except for ELISA test in corticosterone in mice. In roe deer testosterone (T) and cortisol (CORT) were quantify during pre- and post-rut period. Statistical differences were founded between periods, with opposite trends (Hair testosterone pre-rut 1.48 pg/mg and post-rut 2.49 pg/mg, hair cortisol pre-rut 8,72 pg/mg and post-rut 0,49 pg/mg). Progesterone (P4) and CORT were evaluated in red deer. The hair CORT and P4 ranges are respectively 0.03–0.55 pg/mg and 19.47–153.57 ng/mg with no statistical differences. Corticosterone and testosterone (T) hormonal hair profile of laboratory male mice were investigated to verify a potential relationship with age and housing. Hormone levels (6.42 pg/mg for T 502.1 pg/mg for corticosterone, 23.16 ± 7.00 pg/mg for dehydroepiandrosterone) showed a statistical correlation between each other, no statistical differences between the two groups (pair and groups) were founded for T and corticosterone. The concentration of DHEA was statistically affected by the type of housing (p = 0.0377, Student’s t test). These results suggested that such housing practice may be beneficial for social interactions. In the end, in Appennine wolf CORT, T, DHEA, P4, and oestradiol (E2), were assed using radioimmunoassay, but no statistical differences were founded between groups. These results showed to possibility to observe the physiological pathways and animal welfare from analysis from hair in wild or laboratory animals, where it is often difficult to collect other matrices for analysis. [1] A. Elmi et al., «Comparison of hair steroid levels among Wistar rats exposed to different environmental enrichment settings», Vet World, pp. 2731–2735, dic. 2024, doi: 10.14202/vetworld.2024.2731-2735. [2] A. Elmi et al., «Testicular Melatonin and Its Pathway in Roe Deer Bucks (Capreolus capreolus) during Pre- and Post-Rut Periods: Correlation with Testicular Involution», Animals, vol. 11, fasc. 7, p. 1874, giu. 2021, doi: 10.3390/ani11071874. [3] A. Elmi et al., «Quantification of Hair Corticosterone, DHEA and Testosterone as a Potential Tool for Welfare Assessment in Male Laboratory Mice», Animals, vol. 10, fasc. 12, p. 2408, dic. 2020, doi: 10.3390/ani10122408. [4] D. Ventrella et al., «Progesterone and Cortisol Levels in Blood and Hair of Wild Pregnant Red Deer (Cervus Elaphus) Hinds», Animals, vol. 10, fasc. 1, p. 143, gen. 2020, doi: 10.3390/ani10010143. [5] L. A. Frank, K. A. Hnilica, e J. W. Oliver, «BlackwellPublishing,Lt Ad. drenal steroid hormone concentrations in dogs with hair cycle arrest (Alopecia X ) before and during treatment with melatonin and mitotane», Veterinary Dermatology, 2004. [6] J. Wojtaś, «Hair cortisol levels in cats with and without behavioural problems», Journal of Feline Medicine and Surgery, vol. 25, fasc. 2, p. 1098612X221150624, feb. 2023, doi: 10.1177/1098612X221150624.