In red female population (F), in blue male population (M), in green freemartin population (FM)

In red female population (F), in blue male population (M), in green freemartin population (FM). analysis providing robust bases for objective tissue screening, especially in the field of neurodegenerative pathologies. Electronic supplementary material The online version of this article (10.1007/s00429-020-02147-x) contains supplementary material, which is available to authorized users. can be a proper A-366 candidate (Peruffo et al. 2014). Their gestation period (41?weeks) is comparable to the human pregnancy (38C40?weeks), and their brain is large and highly convoluted (Ballarin et al. 2016). The key factor in favor of this model is that bovine frequently shows naturally occurring intersex calves, due to the freemartin syndrome. This condition occurs following the formation of vascular connections between the A-366 placentas of heterosexual twin fetuses and disturbs the sex differentiation of the female twin via the anti-Mllerian hormone production (Rota et al. 2002; Cabianca et al. 2007). Visible consequences on freemartin heifers include body masculinization (Gregory et al. 1996), dramatic changes in the reproductive tract and failure to enter estrus (Marcum 1974; Long 1990; Padula 2005). In this context, the intersex bovine freemartin offers an interesting model to study sex differences of the brain and development in translational medicine (Gra?c et al. 2018). Furthermore, a previous in A-366 vitro study performed on this species in our laboratory reported that granule cells of the female cerebellum showed significantly larger morphological values than the corresponding male elements (Montelli et al. 2017). Since the cerebellum offers a good model to develop computational statistical approaches to the study of single cell morphology, we A-366 studied the structure of vermal lobules VIII and IX of male, female and intersex freemartins bovines. The present study aims at providing clarification on controversial results in sex-related cerebellar differences while acknowledging the freemartin syndrome as a valuable intersex animal model. In addition, this multivariate and multi-aspect method can be extended to study virtually any brain region, providing a robust base for tissue screening, including for the presence of neurodegenerative features. Materials and methods Tissue sampling A series of 28 adult bovine brains (10 males, 10 females and 8 freemartins, all 24?months old), were obtained from local abattoirs in the Veneto region. Animals were treated according to the present European Community Council directive concerning animal welfare during the commercial slaughtering process and were constantly monitored under mandatory official veterinary medical care. The cerebella were collected under sterile conditions and fixed by immersion in phosphate-buffered formaldehyde 4% for 1?month. From each cerebellum, the lobules VIII and IX, classical paleocerebellar lobules located at the postero-inferior part of the vermis, were sampled, re-immersed in buffered formalin, then washed in phosphate saline buffer (PBS) 0.1?M, pH 7.4 and processed for paraffin embedding. Nissl staining The lobules VIII and IX of each specimen were cut into 8-m-thick parasagittal sections. For each cerebellar sample, one section every five was stained (a total of 10 slides per individual per sex). Sections were stained following a standard Nissl protocol: sections were deparaffinized in xylene for 3??5?min, followed by a hydration A-366 series in graded alcohols for 3?min each. After 3?min in distilled water, sections were Rabbit Polyclonal to Tau stained in 0.1% cresyl violet solution for 10?min at 57?C. Sections were then differentiated in 95% alcohol for 20?min. After rinsing in distilled water, sections followed an ascending series of dehydration in graded alcohols for 3?min each, and finally 3??5?min in xylene. The sections were then covered with mounting medium and coverslip glass. The most recent anatomical description of the bovine brain (Okamura 2002) contains illustrations of coronal sections including the main features of the subcortex. Additional details can be found in Yoshikawa (1968). The gross anatomy of the cerebellum was assessed using these references and.