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In an effort to systematically describe the neurochemical anatomy of the bovine anterior hypothalamus, we used a series of immunocytochemical markers such as acetylcholine esterase (AChE), arginine-vasopressin (AVP), calbindin (Calb), galanin (Gal), neuropeptide-Y (NPY), oxytocin (OXT), somatostatin (SST), and vasoactive intestinal peptide (VIP). We also investigated the potential sex difference present in the suprachiasmatic nucleus (SCN) and the vasopressin-oxytocin containing nucleus (VON) of six male and six female Bos taurus. Our study revealed that the cytochemical structure of the cattle anterior hypothalamus follows the blueprint of other mammals. The VON, which was never described before in cattle, showed a sex difference with a 33.7% smaller volume and 23.2% fewer magnocellular neurons (approximately 20-30 μm) in the male. The SCN also did show a sex difference in VIP neurons and volume with a 36.1% larger female nucleus with 28.1% more cells. Additionally, we included five heifers with freemartin syndrome as a new animal model relevant to sexual differentiation in the brain. This is, to the best of our knowledge, the first freemartin study in relation to the brain. Surprisingly, the SCN of freemartin heifers was 32.5% larger than its control male and female counterparts with 29% more VIP cells. Conversely, the freemartin VON had an intermediary size between male and female. To analyze our data, a classical statistical analysis and a novel multivariate and multi-aspect approach were applied. These findings shed new light on sexual dimorphism in the bovine brain and present this species with freemartins as a valuable animal model in neuroscience., (© 2018 Wiley Periodicals, Inc.)

In the mammalian brain, the differentiation of neural cells and the developmental organization of the underlying circuitry are influenced by steroid hormones. The estrogen 17-β estradiol (E2) is one of the most potent regulators of neural growth during prenatal life, synthetized locally from steroid precursors including prenatal testicular testosterone. Estradiol promotes brain differentiation counting sexually dimorphic neural circuits by binding to the estrogen receptors, ER-α and ER-β. The cerebellum has been described as a site of estrogen action and a potentially sexually dimorphic area. The goal of this study was to analyze the capacity of E2 to affect the growth of male and female fetal bovine cerebellar granule. We performed primary cultures of fetal cerebellar granules, and verified the mRNA expression of the ER-α and ER-β in both sexes. Moreover, the distribution of ERs in the male and female cerebellar granules of the second fetal stage was characterized by immunohistochemistry. We measured morphological parameters in presence (or absence) of estradiol administration, focusing on the variations of the dendritic branching pattern of granule neurons. By using the nonparametric combination and permutation testing approach, we proposed a sophisticated multivariate statistical analysis to demonstrate that E2 induces multifarious and dimorphic changes in the granule cells. E2 exerts trophic effects in both female and male granules and this effect is stronger in female. Male granules treated with E2 became similar to female control granule. Bos taurus species has a long gestation and a large brain that offers an interesting alternative in comparative neuroscience., (© 2016 S. Karger AG, Basel.)

The domestic bovine Bos taurus is raised worldwide for meat and milk production, or even for field work. However the functional anatomy of its central nervous system has received limited attention and most of the reported data in textbooks and reviews are derived from single specimens or relatively old literature. Here we report information on the brain of Bos taurus obtained by sampling 158 individuals, 150 of which at local abattoirs and 8 in the dissecting room, these latter subsequently formalin-fixed. Using body weight and fresh brain weight we calculated the Encephalization Quotient (EQ), and Cerebellar Quotient (CQ). Formalin-fixed brains sampled in the necropsy room were used to calculate the absolute and relative weight of the major components of the brain. The data that we obtained indicate that the domestic bovine Bos taurus possesses a large, convoluted brain, with a slightly lower weight than expected for an animal of its mass. Comparisons with other terrestrial and marine members of the order Cetartiodactyla suggested close similarity with other species with the same feeding adaptations, and with representative baleen whales. On the other hand differences with fish-hunting toothed whales suggest separate evolutionary pathways in brain evolution. Comparison with the other large domestic herbivore Equus caballus (belonging to the order Perissodactyla) indicates that Bos taurus underwent heavier selection of bodily traits, which is also possibly reflected in a comparatively lower EQ than in the horse. The data analyzed suggest that the brain of domestic bovine is potentially interesting for comparative neuroscience studies and may represents an alternative model to investigate neurodegeneration processes.

The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of "deceleration aging factor" as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models.

Statistical tests in medical research: traditional methods vs. multivariate npc permutation tests.Within medical research, a useful statistical tool is based on hypotheses testing in terms of the so-called null, that is the treatment has no effect, and alternative hypotheses, that is the treatment has some effects. By controlling the risks of wrong decisions, empirical data are used in order to possibly reject the null hypotheses in favour of the alternative, so that demonstrating the efficacy of a treatment of interest. The multivariate permutation tests, based on the nonparametric combination - NPC method, provide an innovative, robust and effective hypotheses testing solution to many real problems that are commonly encountered in medical research when multiple end-points are observed. This paper discusses the various approaches to hypothesis testing and the main advantages of NPC tests, which consist in the fact that they require much less stringent assumptions than traditional statistical tests. Moreover, the related results may be extended to the reference population even in case of selection-bias, that is non-random sampling. In this work, we review and discuss some basic testing procedures along with the theoretical and practical relevance of NPC tests showing their effectiveness in medical research. Within the non-parametric methods, NPC tests represent the current "frontier" of statistical research, but already widely available in the practice of analysis of clinical data.

Microclimatic conditions were recorded in an Italian school and Fanger's indexes PMV and PPD were calculated under different conditions. Students' sensations were investigated four times by means of two surveys, one related to actual microclimatic conditions and one on overall satisfaction, interaction occupant-building and reactions to discomfort. Pupils' classroom position was considered to look for possible influence on thermal comfort: a difference emerged from PMV and the survey, but the results obtained from the two approaches differ for both the entity of discomfort and its distribution within each classroom. Innovative multivariate nonparametric statistical techniques were applied to compare and rank the classrooms in accordance with students' subjective perceptions; a global ranking has been also calculated, considering thermal and visual comfort and air quality. Comparing pupil-sensation-based ranking with environmental parameters no clear correspondence was found, except for mid-season, where PMV, CO2 concentration and desk illuminance were similar in all the classrooms., (Copyright © 2014 Elsevier Ltd and The Ergonomics Society. All rights reserved.)

Estrogens diversely affect various physiological processes by genomic or non-genomic mechanisms, in both excitable and non-excitable cells. Additional to the trophic effects of estrogens promoting cell growth and differentiation, recent experimental evidence highlights their involvement in the regulation of intracellular Ca(2+) homeostasis. The effects of estrogens on excitable cells are well documented. However, these steroids also influence numerous physiological events in non-excitable cells, such as fibroblasts or vascular endothelial cells. We have focused our attention on an immortalized endothelial-like cell line derived from fetal bovine cerebellum. Estradiol (E(2)) effects on intracellular Ca(2+) homeostasis were tested by varying the exposure time to the hormone (8, 24, 48 h). Calcium measurements were performed with genetically encoded Ca(2+) probes (Cameleons) targeted to the main subcellular compartments involved in intracellular Ca(2+) homeostasis (cytosol, endoplasmic reticulum, mitochondria). Mitochondrial Ca(2+) uptake significantly decreased after 48-h exposure to E(2), whereas cytosolic and endoplasmic reticulum responses were unaffected. The effect of E(2) on mitochondrial Ca(2+) handling was blocked by ICI 182,780, a pure estrogen receptor antagonist, suggesting that the effect was estrogen-receptor-mediated. To evaluate whether the decrease of Ca(2+) uptake affected mitochondrial membrane potential (ΔΨm), cells were monitored in the presence of tetra-methyl-rhodamine-methylester; no significant changes were seen between cells treated with E(2) and controls. To investigate a mechanism of action, we assessed the possibile involvement of the permeability transition pore (PTP), an inner mitochondrial membrane channel influencing energy metabolism and cell viability. We treated cells with CyclosporinA (CsA), which binds to the matrix chaperone cyclophilin-D and regulates PTP opening. CsA reversed the effects of a 48-h treatment with E(2), suggesting a possible transcriptional modulation of proteins involved in the mitochondrial permeability transition process.

The aim of this study was to apply biometric measurements and analyses of proximate composition, fatty acid composition, and ratios of stable isotopes of carbon (δ(13)C) and nitrogen (δ(15)N) in muscle tissue to reliably differentiate between wild and farmed European sea bass (Dicentrarchus labrax). Farmed (n = 20) and wild (n = 19) European sea bass were purchased between March and May 2008 and used as standard samples. In the same months, a survey was conducted to evaluate the truthfulness of the statements on the labels of European sea bass sold in retail markets (declared farmed n = 34 and declared wild n = 33). In addition, data from the literature (reference) were employed to build the profile type of wild and farmed European sea bass. Primarily, an exploration and comparison of the analytical data of the standard data set based on principal component analysis and permutation test were performed. Afterward, an inferential statistical approach based on nonparametric combination test methodology (NPC) was applied on standard samples to check its suitability in discriminating the production method. This multivariate statistical analysis selected 30 variables on a total of 36 available. The validation of standard fish data set was accomplished by a novel nonparametric rank-based method according to profile type (just 1 misclassification over 39 samples). Both the NPC test and nonparametric rank-based method were then applied to survey fishes using the selected variables with the aim to classify the individual European sea bass as "true farmed" or "true wild". The former test segregated 10 fishes over 33 declared wild, whereas the results obtained by the nonparametric rank-based method showed that 11 of 33 declared wild European sea bass samples could be unquestionably attributed to the wild cluster. Moreover, considering the comparative contribution of profile type, a few surveyed farmed samples were ascribed to the wild cluster.

Background and Aim of the Study: Historically, tricuspid valve replacement (TVR) has been associated with high mortality and morbidity, and current knowledge in the long-term outcome of TVR is limited. The study aim was to review the authors' experience from a consecutive series of patients., Methods: Between January 1990 and December 2005, a total of 43 patients (seven males, 36 females; mean age 52 +/- 14 years) underwent TVR. The etiology was rheumatic in 33 patients (77%) and degenerative disease in 10 (22%). Thirty-six patients (84%) were in NYHA class III or IV. Thirty-four patients (79%) underwent redo procedures; all patients underwent TVR with a mechanical prosthesis., Results: The overall operative mortality was 16% (n = 7). Of the 36 survivors, nine (25%) died during follow up. The Kaplan-Meier survival at 2.5, 5, and 10 years was 78%, 70%, and 58%, respectively. Five patients (14%) underwent reoperation during follow up (three for tricuspid valve thrombosis) and all five survived the reoperation. Freedom from reoperation at five and 10 years was 90% and 74%, respectively. On permutation test analysis, older age, liver congestion and redo surgery were found to be the major determinants of long-term mortality., Conclusion: TVR carries a higher short- and long-term mortality when compared to left-heart valve surgery. A timely referral before the development of end-stage cardiac impairment might determine a further improvement in outcome.

The determination of age is an important step in defining the life history traits of individuals and populations. Age determination of odontocetes is mainly based on counting annual growth layer groups in the teeth. However, this useful method is always invasive, requiring the cutting of at least one tooth, and sometimes the results are difficult to interpret. Based on the concept that bone matrix is constantly deposited throughout life, we analysed the bone mineral density of the arm and forearm of a series of bottlenose dolphins (Tursiops truncatus, Montagu 1821) stranded along the Italian coast of the Adriatic Sea or maintained in confined waters. The bone mineral density values we obtained were evaluated as possible age predictors of the Mediterranean population of this species, considering age as determined by counting growth layer groups in sections of the teeth and the total body length of the animal as references. Comparisons between left and right flipper showed no difference. Our results show that bone mineral density values of the thoracic limb are indeed reliable age predictors in Tursiops truncatus. Further investigations in additional odontocete species are necessary to provide strong evidence of the reliability of bone mineral density as an indicator of growth and chronological wear and tear in toothed-whales.