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ABSTRACT: Heat stress (HS) is one of the pivotal causes of economic losses in dairy industries and affects welfare and performance, but its effect on milk microbiota remains elusive. It is also unclear if and how different breeds may cope with HS in sustaining productive performance. The objectives of this study were to compare (1) the performance of 2 dairy breeds, namely Holstein and Brown Swiss, subjected to HS and (2) the different effects of HS on the milk microbiota of the 2 breeds in thermal comfort conditions and HS. The study was carried out on 36 dairy cows, 18 per breed. The HS was induced by switching off the cooling system during a natural heat wave for 4 d. In addition to the temperature-humidity index, animal stress was confirmed by measuring respiratory frequency and rectal temperature twice daily at 0400 h and 1500 h. The HS affected the 2 breeds differently. The rectal temperature increased with HS in both breeds. Milk yield recording and sampling were performed during the morning milking of d 1 (at 0400 h) and afternoon milking of d 4 (at 1700 h). Productive parameters were also different: milk yield, FCM, ECM, protein and casein content, and renneting parameters were decreased in Holstein cows but remained unaffected in Brown Swiss cows. The HS also modified the milk microbiota of the 2 breeds differently. During HS, the Brown Swiss cows had milk microbiota that was richer (α diversity) than that of the Holstein cows. Comparing the time points before and during HS within breeds showed that Brown Swiss cow milk microbiota was less affected by HS than Holstein cow milk microbiota. Under the same thermal comfort condition, milk microbiota did not discriminate between Brown Swiss and Holstein. Consistently with α and β diversity, the number of operational taxonomic units (OTU) at the genus level that changed their abundance during HS was higher in Holstein (74 OTU) than in Brown Swiss (only 20 OTU). The most significant changes in abundance affected Acinetobacter, Chryseobacterium, Cutibacterium, Enterococcus, Lactococcus, Prevotella-9, Serratia, and Streptococcus. In conclusion, the present report confirms and extends previous studies by demonstrating that Brown Swiss cows regulate their body temperature better than the Holstein breed. The relative thermal tolerance to HS compared with Holstein cows is also confirmed by changes in milk uncultured microbiota, which were more evident in Holstein cows than in Brown Swiss cows.

Abstract The impact of nutritional modification to increase functional polyunsaturated fatty acids (PUFA), such as n-3 and n-6 fatty acids (FA) or conjugated linoleic acid (CLA), on milk proteome profile during early lactation remains largely unknown. We used an untargeted proteomics approach to investigate the impact of lactation day and PUFA supplementation on the proteome signature in skimmed milk over the course of early lactation. Sixteen Holstein dairy cows received abomasal infusion of saturated FA (CTRL) or a mixture of essential FA and CLA (EFA + CLA group) from − 63 to + 63 days relative to parturition. Using quantitative proteomics, 479 unique proteins were identified in skimmed milk at days 1, 28, and 63 postpartum. The top discriminating proteins between transition milk (day 1) and mature milk (days 28 and 63), including members of complements (i.e. C2 and C5), growth factor (TGFB2), lipoproteins (i.e. APOE and APOD), and chaperones (i.e. ST13 and CLU), are associated with calves’ immune system and gut development. The EFA + CLA supplementation moderately affected a few proteins associated with regulating mammary glands’ lipogenesis through the (re)assembly of lipoprotein particles, possibly under the PPAR signaling pathway. Collectively, skimmed milk proteome is dynamically regulated initially by cow’s metabolic and physiological changes and to a lesser extent by nutritional PUFA modifications.

Background: Physical Education (PE) classes are vital for nurturing students' social development and promoting collaboration. This study examined how primary and middle school students perceived PE classes, with a focus on collaboration, well-being and enjoyment, teacher behavior, the inclusion of nondisabled and disabled students, and general class behavior. Methods: One-hundred and seventy students (24 primary school and 146 middle school students) were surveyed using a questionnaire. Results: Exploratory Factor Analysis identified seven factors including: well-being, activities for all students, teacher-oriented behavior, collaboration, and attention to students with disabilities. Linear regressions on each identified Factor revealed higher well-being (Factor 1) between primary and middle school students. In addition, the perceived well-being in PE classes and staying together with classmates among younger middle school students was found to be higher than that reported by their older counterparts in middle school. The analysis also revealed that males perceived a higher well-being in PE classes than females. Conclusion: The research underscores that primary and especially middle school PE teachers should promote the well-being of students by creating inclusive and enjoyable PE classes. The PE classes should consider the differences between sexes and the individual differences. Finally, this research lays the foundation for future investigations to refine inclusive PE strategies and improve students' well-being.

ABSTRACT: Citrus pectin (CP) is a dietary fiber used in animal nutrition with anti-inflammatory properties. CP downregulates chicken immunoregulatory monocytes' functions, like chemotaxis and phagocytosis, in vitro. The molecular underlying background is still unknown. This study investigated the activity of CP on chicken peripheral blood mononuclear cells (PBMC) proteome. An overall number of 1503 proteins were identified and quantified. The supervised sparse variant partial least squares—discriminant analysis (sPLS-DA) for paired data highlighted 373 discriminant proteins between CP-treated and the control group, of which 50 proteins with the highest abundance in CP and 137 in the control group were selected for Gene Ontology (GO) analyses using ProteINSIDE. Discriminant Protein highly abundant in CP-treated cells were involved in actin cytoskeleton organization and negative regulation of cell migration.Interestingly, MARCKSL1, a chemotaxis inhibitor, was upregulated in CP-treated cells. On the contrary, CP incubation downregulated MARCKS, LGALS3, and LGALS8, which are involved in cytoskeleton rearrangements, cell migration, and phagocytosis. In conclusion, these results provide a proteomics background to the anti-inflammatory activity of CP, demonstrating that the in vitro downregulation of phagocytosis and chemotaxis is related to changes in proteins related to the cytoskeleton.

Abstract Controlling foodborne pathogens in buffalo milk is crucial for ensuring food safety. This study estimated the prevalence of nine target genes representing seven critical foodborne bacteria in milk and milk products, and identified factors associated with their presence in buffalo milk chain nodes in Bangladesh. One hundred and forty-three milk samples from bulk tank milk (n = 34), middlemen (n = 37), milk collection centers (n = 37), and milk product shops (n = 35) were collected and analyzed using RT-PCR. Escherichia (E.) coli, represented through yccT genes, was the most prevalent throughout the milk chain (81–97%). Chi-squared tests were performed to identify the potential risk factors associated with the presence of foodborne bacteria encoded for different genes. At the middleman level, the prevalence of E. coli was associated with the Mymensingh, Noakhali, and Bhola districts (P = 0.01). The prevalence of Listeria monocytogenes, represented through inlA genes, and Yersinia (Y.) enterocolitica, represented through yst genes, were the highest at the farm level (65–79%). The prevalence of both bacteria in bulk milk was associated with the Noakhali and Bhola districts (P

Essential oils (EOs) are aromatic compounds known for their potent anti-inflammatory and antibacterial properties. However, these molecules are susceptible to degradation when exposed to external conditions, such as light and oxygen. Numerous studies have reported the protective effects of encapsulation on sensitive bioactive, such as EOs, for improved or preserved stability and shelf life. The aim of this study was to investigate the antibacterial activity of different formulations of carvacrol, the most important and used component of EOs. We comparatively evaluated the activity of free, protein-complexed and nano-emulsified carvacrol against two selected pathogenic bacteria: Staphylococcus aureus (a Gram+ strain) and Escherichia coli (a Gram- strain). Free carvacrol exhibited antibacterial activity against both tested bacteria, with comparable minimum inhibitory concentrations of 380 µg/mL for S. aureus and 370 µg/mL for E. coli. Indeed, carvacrol interact strongly with lactoferrin and β-lactoglobulin, two model carrier proteins, with preserved antibacterial activity. Unexpectedly, nano-emulsification of carvacrol, resulting in nano-droplets of 100 to 240 nm, decreased its antibacterial effect. Therefore, complexation with food proteins is a viable option to formulate a carrier with preserved carvacrol bioactivity. Ongoing studies aim to understand the reasons behind the recovered low activity of nano-emulsified carvacrol on the two pathogen species and under used experimental set-up.

Abstract Exosomes mediate near and long-distance intercellular communication by transferring their molecular cargo to recipient cells, altering their biological response. Milk exosomes (MEx) are internalized by immune cells and exert immunomodulatory functions in vitro. Porcine MEx can accumulate in the small intestine, rich in macrophages. No information is available on the immunomodulatory ability of porcine MEx on porcine monocytes, which are known precursors of gut macrophages. Therefore, this study aims at (1) assessing the in vitro uptake of porcine MEx by porcine monocytes (CD14+), and (2) evaluating the in vitro impact of porcine MEx on porcine monocytes immune functions. MEx were purified by ultracentrifugation and size exclusion chromatography. The monocytes’ internalization of PKH26-labeled MEx was examined using fluorescence microscopy. Monocytes were incubated with increasing exosome concentrations and their apoptosis and viability were measured. Lastly, the ability of MEx to modulate the cells’ immune activities was evaluated by measuring monocytes’ phagocytosis, the capacity of killing bacteria, chemotaxis, and reactive oxygen species (ROS) production. MEx were internalized by porcine monocytes in vitro. They also decreased their chemotaxis and phagocytosis, and increased ROS production. Altogether, this study provides insights into the role that MEx might play in pigs’ immunity by demonstrating that MEx are internalized by porcine monocytes in vitro and exert immunomodulatory effects on inflammatory functions.

Abstract Staphylococcus aureus modulates the host immune response directly by interacting with the immune cells or indirectly by secreting molecules (secretome). Relevant differences in virulence mechanisms have been reported for the secretome produced by different S. aureus strains. The present study investigated the S. aureus secretome impact on peripheral bovine mononuclear cells (PBMCs) by comparing two S. aureus strains with opposite epidemiological behavior, the genotype B (GTB)/sequence type (ST) 8, associated with a high within-herd prevalence, and GTS/ST398, associated with a low within-herd prevalence. PBMCs were incubated with different concentrations (0%, 0.5%, 1%, and 2.5%) of GTB/ST8 and GTS/ST398 secretome for 18 and 48 h, and the viability was assessed. The mRNA levels of pro- (IL1-β and STAT1) and anti-inflammatory (IL-10, STAT6, and TGF-β) genes, and the amount of pro- (miR-155-5p and miR-125b-5p) and anti-inflammatory (miR-146a and miR-145) miRNAs were quantified by RT-qPCR. Results showed that incubation with 2.5% of GTB/ST8 secretome increased the viability of cells. In contrast, incubation with the GTS/ST398 secretome strongly decreased cell viability, preventing any further assays. The GTB/ST8 secretome promoted PBMC polarization towards the pro-inflammatory phenotype inducing the overexpression of IL1-β, STAT1 and miR-155-5p, while the expression of genes involved in the anti-inflammatory response was not affected. In conclusion, the challenge of PBMC to the GTS/ST398 secretome strongly impaired cell viability, while exposure to the GTB/ST8 secretome increased cell viability and enhanced a pro-inflammatory response, further highlighting the different effects exerted on host cells by S. aureus strains with epidemiologically divergent behaviors.

Abstract Bubaline alphaherpesvirus 1 (BuHV-1) is a pathogen of water buffaloes responsible for economic loss worldwide. MicroRNAs (miRNAs) regulate gene expression produced by alphaherpesviruses and hosts. This study aimed at (a) unravelling the ability of BuHV-1 to produce miRNAs, including hv1-miR-B6, hv1-miR-B8, hv1-miR-B9; (b) measuring the host immune-related miRNAs associated to herpesvirus infection, including miR-210-3p, miR-490-3p, miR-17-5p, miR-148a-3p, miR-338-3p, miR-370-3p, by RT-qPCR; (c) identifying candidate markers of infection by receiver-operating characteristic (ROC) curves; (d) exploiting the biological functions by pathway enrichment analyses. Five water buffaloes BuHV-1 and Bovine alphaherpesvirus 1 (BoHV-1) free were immunized against Infectious Bovine Rhinotracheitis (IBR). Five additional water buffaloes served as negative controls. All animals were challenged with a virulent wild-type (wt) BuHV-1 via the intranasal route 120 days after the first vaccination. Nasal swabs were obtained at days (d) 0, 2, 4, 7, 10, 15, 30, and 63 post-challenge (pc). The animals of both groups shed wt BuHV-1 up to d7 pc. Results demonstrated that (a) miRNAs produced by the host and BuHV-1 could be efficiently quantified in the nasal secretion up to d63 and d15 pc, respectively; b) the levels of host and BuHV-1 miRNAs are different between vaccinated and control buffaloes; c) miR-370-3p discriminated vaccinated and control animals; d) host immune-related miRNAs may modulate genes involved in the cell adhesion pathway of the neuronal and immune system. Overall, the present study provides evidence that miRNAs can be detected in nasal secretions of water buffaloes and that their expression is modulated by BuHV-1.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Abstract An in vitro trial was carried out to investigate the effects of natural Thymbra capitata essential oil (NEO) and its main compounds [including carvacrol, p-cymene, γ-terpinene given alone or in a synthetic combination (SEO)] on ruminal fermentation and the bacterial community using batch cultures inoculated with ruminal digesta and incubating two different basal diets [high-forage (F) and high-concentrate (C) diet]. After 24 h of incubation, primary fermentation end-products [gas, methane, volatile fatty acids (VFAs) and ammonia] and rumen microbial diversity were determined. NEO reduced the total VFA concentration (P 0.05) by any of the experimental treatments or diets evaluated. Microbial diversity analysis showed only a moderate effect of carvacrol and SEO on 13 genera, including, mainly, Atopobium and Blautia (involved in subacute ruminal acidosis) or Candidatus Saccharimonas (related to laminitis). In conclusion, T. capitata EO has a limited potential to attain nutritional or environmental benefits, but further research should be carried out to clarify its effects on animal health and microbial food safety.

The goal of this study was to determine the plasma concentrations of the acute phase proteins fibrino-gen and ceruloplasmin during the pregnancy and postpartum period in goats. The experiment was carried out with six clinically healthy Bulgarian native goats, aged 46 years, weighing 4956 kg, reared at the Biobase of the Faculty of Veterinary Medicine – Stara Zagora. Blood samples for assay of plasma concentrations of the studied acute-phase proteins during pregnancy were collected over 7-day intervals, from the 14th to the 147th day of pregnancy. During the postpartum period, samples were obtained at 3-day intervals between the 1st and 15th postpartum day, as well as once on the 20th and 30th day after the parturition. The results indicated that throughout the first trimester of the preg-nancy, the highest average plasma fibrinogen values were established on the 14th day, while signifi-cantly lower values (P

Abstract A growing body of evidence shows that microRNA (miRNA), play important roles in regulating adipose tissue (AT) physiology and function. The objective was to characterize the AT miRNA profile in over-conditioned (HBCS, n = 19) versus moderate-conditioned (MBCS, n = 19) periparturient dairy cows. Tail-head subcutaneous AT biopsied on d -49 and 21 relative to parturition were used for miRNA sequencing. The miR-486 was the most significant miRNA among the upregulated miRNA on d -49, which might be related to more pronounced changes in lipogenesis and altered insulin sensitivity in AT of HBCS cows at dry-off. Comparing HBCS to MBCS on d 21, 23 miRNA were downregulated and 20 were upregulated. The predicted targets of upregulated differentially expressed (DE)-miRNA on d 21 were enriched in different pathways, including pathways related to lysosomes and peroxisomes. The predicted targets of downregulated DE-miRNA on d 21 were enriched in various pathways, including epidermal growth factor receptor, insulin resistance, hypoxia-inducible factor 1 signaling pathway, and autophagy. The results showed that over-conditioning was associated with changes in SCAT miRNA profile mainly on d 21, of which most were downregulated. The enriched pathways may participate in over-conditioning-associated metabolic challenges during early lactation.

Cows fed total mixed rations (silage-based) may not receive as much essential fatty acids (EFAs) and conjugated linoleic acids (CLAs) as cows fed pasture-based rations (fresh grass) containing rich sources of polyunsaturated fatty acids. CLA-induced milk fat depression allows dairy cows to conserve more metabolisable energy, thereby shortening the state of negative energy balance and reducing excessive fat mobilisation at early lactation. EFAs, particularly α-linolenic acid, exert anti-inflammatory and antioxidative properties, thereby modulating immune functions. Thus, combined EFA and CLA supplementation seems to be an effective nutritional strategy to relieve energy metabolism and to improve immune response, which are often compromised during the transition from late pregnancy to lactation in high-yielding dairy cows. There has been extensive research on this idea over the last two decades, and despite promising results, several interfering factors have led to varying findings, making it difficult to conclude whether and under what conditions EFA and CLA supplementations are beneficial for dairy cows during the transition period. This article reviews the latest studies on the effects of EFA and CLA supplementation, alone or in combination, on dairy cow metabolism and health during various stages around parturition. Our review article summarises and provides novel insights into the mechanisms by which EFA and/or CLA influence markers of metabolism, energy homeostasis and partitioning, immunity, and inflammation revealed by a deep molecular phenotyping.

Abstract This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

The present study aims to evaluate the changes in concentrations of some acute phase proteins during Haemonchus contortus infection in lambs. This experiment was performed using 12 three-month-old healthy lambs, randomly allocated into 2 equal groups: G1 (uninfected control animals) and G2 (lambs infected with H. contortus). Each lamb of G2 group was orally inoculated with 4000 infective third stage larvae (L3) of H. contortus by placing a probe. Blood samples were collected on post infection days zero, 4, 7, 11, 14, 21, 28 and 32. After sampling, the blood was centrifuged, and the separated plasma was used for the quantitative determination of haptoglobin (Hp) and fibrinogen (Fb). The most pronounced changes occurred in Hp levels, which increased and attained the highest values on post infection days 4, 7, and 11. A peak concentration occurred on post infection day 7 when Hp increased by 45.96% vs the initial level, and by 44.08% vs the control level on the same day. There were no significant changes in Fb concentrations throughout the study.

Abstract Mastitis by non-aureus staphylococci (NAS) is a significant issue in dairy buffalo farming. In a herd with subclinical NAS mastitis, we identified Staphylococcus microti as the predominant species. To assess milk protein integrity and investigate potential disease markers, we characterized 12 NAS-positive and 12 healthy quarter milk samples by shotgun peptidomics combining peptide enrichment and high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS). We observed significant changes in the milk peptidome. Out of 789 total peptides identified in each group, 49 and 44 were unique or increased in NAS-positive and healthy milk, respectively. In NAS-positive milk, the differential peptides belonged mainly to caseins, followed by milk fat globule membrane proteins (MFGMP) and by the immune defense/antimicrobial proteins osteopontin, lactoperoxidase, and serum amyloid A. In healthy milk, these belonged mainly to MFGMP, followed by caseins. In terms of abundance, peptides from MFGMP and immune defense protein were higher in NAS-positive milk, while peptides from caseins were higher in healthy milk. These findings highlight the impact of NAS on buffalo milk quality and mammary gland health, even when clinical signs are not evident, and underscore the need for clarifying the epidemiology and relevance of the different NAS species in this dairy ruminant.

Physical activity in the form of “active breaks” can be combined with academic instruction in primary school. However, few studies have examined the feasibility of conducting active breaks in secondary school. To address this gap, we conducted focus groups (FGs) regarding the implementation of an active breaks (ABs) protocol with 20 teachers and 10 secondary school students. Barriers/facilitators toward the implementation of ABs were classified using grounded theory inductive methods framed by the socio-ecological model. Individual-level factors were instrumental for both teachers and students. Teachers highlighted personal fears and concerns regarding using ABs, while students reported fears related to peer behaviour during the activity. Both teachers and students agreed that ABs can improve cognitive skills and time-on-task behaviour. Teachers articulated concerns related to student behaviour during ABs including possible social exclusion and injury. Students felt that ABs might affect classroom management and interfere with maintaining students’ academic focus. Teachers underscored that ABs required social support from the administration and colleagues. Students felt that ABs could support teachers’ instructional focus and provide them with an energy respite. Collectively, the FGs suggested that environmental limitations could hinder the implementation of ABs. Involving teacher and student feedback during the codesign phase can rationally inform the design of school-based ABs.

Abstract Essential fatty acids (EFA) and conjugated linoleic acids (CLA) are unsaturated fatty acids with immune-modulatory effects, yet their synergistic effect is poorly understood in dairy cows. This study aimed at identifying differentially abundant proteins (DAP) and their associated pathways in dairy cows supplied with a combination of EFA and CLA during the transition from antepartum (AP) to early postpartum (PP). Sixteen Holstein cows were abomasally infused with coconut oil as a control (CTRL) or a mixture of EFA (linseed + safflower oil) and CLA (Lutalin, BASF) (EFA + CLA) from − 63 to + 63 days relative to parturition. Label-free quantitative proteomics was performed on plasma samples collected at days − 21, + 1, + 28, and + 63. During the transition time, DAP, consisting of a cluster of apolipoproteins (APO), including APOE, APOH, and APOB, along with a cluster of immune-related proteins, were related to complement and coagulation cascades, inflammatory response, and cholesterol metabolism. In response to EFA + CLA, specific APO comprising APOC3, APOA1, APOA4, and APOC4 were increased in a time-dependent manner; they were linked to triglyceride-enriched lipoprotein metabolisms and immune function. Altogether, these results provide new insights into metabolic and immune adaptation and crosstalk between them in transition dairy cows divergent in EFA + CLA status.

The present study aimed to evaluate the changes in concentrations of some biochemical parameters, as well as macro and microscopic alterations during Eimeria stiedae infection in rabbits. The experiment was performed using 12 threemonth- old healthy rabbits, randomly allocated into 2 equal groups: G1 (controls, uninfected animals) and G2 (rabbits infected with E. stiedae). Blood samples were collected at time zero (prior to the infection), 6th, 24th, and 48th hours, and also 7th, 14th, 21st, 28th days after the infection. After sampling, the blood was centrifuged, plasma was separated and frozen at -20 ºC until analyzed. Thawed plasma was used for the quantitative determination of haptoglobin (Hp), total protein (TP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), total cholesterol (TC), total bilirubin (TBIL), urea, and creatinine (CREA). The results in infected rabbits revealed a significant increase in Hp, AST, ALT, GGT, TBIL, and TC levels, as well as a significant decrease in ALP and urea. A weak hyperproteinemia was also observed. There were no changes in CREA concentration. At the end of the clinical investigation, all rabbits were humanely euthanized and necropsied. The post-mortem examination of the infected group revealed hepatomegaly, multifocal yellowish nodules diffusely spread over the liver surface and in the parenchyma, considerably dilated bile ducts, and biliary hyperplasia. Given the results obtained from this experiment, it can be affirmed that hepatic eimeriosis in rabbits is a severe parasitic disease leading to significant disturbances of liver histology and resulting changes in the biochemical profile of infected rabbits.

The need for movement, which characterizes the current generations of children, has become urgent and worrying. Despite the continuous projects carried out in primary school, few seems to improve health and motivation to adopt active lifestyles. Among the many projects carried out in recent years, active breaks seem to be gaining ground for their easiness of realization and sustainability over time. The active breaks proved to be very effective towards well being and cognitive activation representing an additional dose of physical activity that can help children in meeting the recommended levels of daily physical activity. Moreover, the active breaks represent an innovative strategy to improve the classroom climate and facilitate the educational action of teachers. Teachers, involved in the I-MOVE and ABS studies, of an inter-university study group (University of Bologna and University of Roma Tre), responded with great enthusiasm to the practice of active breaks. Teachers highlighted that active break produced a positive effect toward school routine, learning environment and activation of cognitive functions in children. The present paper reports data related to the awareness of teachers, with regard to the application of active breaks in their school day, in terms of feasibility, management and organization in the face of effects considered absolutely positive for the daily school experience.

After years of work, interrupted by plateaus of political pondering, the 2002 Budget Law (Art. 103) has finally officialized the inclusion of specialist teachers for the subject of Physical Education. That is, after the Italian Senate Bill S 992/2020, which mandated the introduction of specialist teachers to guarantee «a real and qualified teaching for children through interventions that are adequate and targeted in terms of motor development, but also to impact learning, prevention, and socialization» (D’Elia, 2022, p. 82).

Abstract Background The World Health Organization stated an average of 60 min of Moderate to Vigorous Physical Activity (MVPA) that children should accumulate every day. Nevertheless physical inactivity is growing and, due to restrictions imposed during pandemic, PA levels of children might be more negatively affected. The study aimed to analyse the impact of COVID-19 on the PA of an Italian sample of primary school children by comparing it before and during COVID-19 considering gender differences. Methods A pre-post analysis (October 2019–January 2021) was conducted using a randomized sample (N = 77) from the I-MOVE study settled in an Italian primary school. Both objective (Actigraph accelerometers) and self-reported (PAQ-c questionnaires) assessments of PA were performed. Changes were compared using T-Student and Chi-Square test. Gender differences were calculated using Anova. Results Weekly and daily minutes time spent in MVPA significantly decreased respectively by − 30.59 ± 120.87 and − 15.32 ± 16.21 from before to during pandemic while the weekly time spent in sedentary behaviour increased (+ 1196.01 ± 381.49). PAQ-c scores followed the same negative trend (− 0.87 ± 0.72). Boys seem to have suffered more than girls from the imposed restrictions. Conclusion These findings outline the need for strategies to promote PA and reduce sedentary behaviours in children to prevent COVID-19 restriction long-term effects.

The Montessorian proposal for childhood education appears highly modern and relevant in relation to the development of both motor skills and cognitive functions (Shivji, 2016;), strongly supported by neurosciences’ embodied theories (Roessingh, H. & Bence, M. 2018)), and the increasing wellbeing problem related to childhood (Pate et al, 2014; Ross, 2012). This review analyses Maria Montessori’s modern educational vision, in light of the emerging needs of today’s children. The contribution reviews existing literature focusing on body and movement, but connected with cognitive, emotional and well-being aspects, which are critical in preschool education, both for educators/teachers (Atli, 2016; Akkerman, 2014; Lillard, 2011), and for school reform policies (Lillard, 2019).

Small extracellular vesicles (sEV) are a class of extracellular vesicles (30–150 nm), delivering molecules including proteins, metabolites, and microRNAs (miRNAs), involved in physiological intercellular crosstalk and disease pathogenesis. The present pilot study aims are (I) to develop an easy and fast protocol for the isolation of sEV from plasma of mast cell tumor (MCT)-affected dogs; (II) to evaluate if miR-21-5p (sEV-miR-21-5p), a miRNA overexpressed by MCT, is associated with sEV. Seventeen dogs have been enrolled in the study: 4 healthy and 13 (6 with and 7 without nodal metastasis) MCT-affected dogs. sEV were isolated using size exclusion chromatography (SEC) (IZON column 35nm) and were characterized by Western blot, Nanoparticle tracking analysis, and transmission electron microscopy. sEV-miR-21-5p was quantified using digital PCR. sEV expressed the specific markers CD9 and TSG101, and a marker of mast cell tryptase. The sEV mean concentration and size were 2.68E + 10 particles/ml, and 99.6 nm, 2.89E + 10 particles/ml and 101.7 nm, and 3.21E + 10 particles/ml and 124 nm in non-metastatic, nodal metastatic, and healthy samples, respectively. The comparative analysis demonstrated that the level of sEV-miR-21-5p was significantly higher in dogs with nodal metastasis compared to healthy (P = 0.038) and without nodal metastasis samples (P = 0.007). In conclusion, the present work demonstrated that a pure population of sEV can be isolated from the plasma of MCT-affected dogs using the SEC approach and that the level of sEV-miR-21-5p is higher in nodal metastatic MCT-affected dogs compared with healthy and MCT-affected dogs without nodal involvement.

In the current study, we investigated dairy cows’ circulating microRNA (miRNA) expression signature during several key time points around calving, to get insights into different aspects of metabolic adaptation. In a trial with 32 dairy cows, plasma samples were collected on days −21, 1, 28, and 63 relative to calving. Individually extracted total RNA was subjected to RNA sequencing using NovaSeq 6,000 (Illumina, CA) on the respective platform of IGA Technology Services, Udine, Italy. MiRDeep2 was used to identify known and novel miRNA according to the miRbase collection. Differentially expressed miRNA (DEM) were assessed at a threshold of fold-change > 1.5 and false discovery rate < 0.05 using the edgeR package. The MiRWalk database was used to predict DEM targets and their associated KEGG pathways. Among a total of 1,692 identified miRNA, 445 known miRNA were included for statistical analysis, of which 84, 59, and 61 DEM were found between days −21 to 1, 1 to 28, and 28 to 63, respectively. These miRNA were annotated to KEGG pathways targeting the insulin, MAPK, Ras, Wnt, Hippo, sphingolipid, T cell receptor, and mTOR signaling pathways. MiRNA-mRNA network analysis identified miRNA as master regulators of the biological process including miR-138, miR-149-5p, miR-2466-3p, miR-214, miR-504, and miR-6523a. This study provided new insights into the miRNA signatures of transition to the lactation period. Calving emerged as a critical time point when miRNA were most affected, while the following period appeared to be recovering from massive parturition changes. The primarily affected pathways were key signaling pathways related to establishing metabolic and immune adaptations.

The aim of this study was to assess the effect of nucleotides administration on growth performance and immune response in post-weaning piglets. Twenty-eight male weaned piglets, homogeneous for age and weight were randomly allocated to two experimental treatments. Treated group (T) was daily orally administered 0.8 g/head of a mixture of nucleotides suspended in 2.1 mL water solution; while control group (C) received 2.1 mL saline solution. Body weight (BW) and average daily gain (ADG) were individually recorded weekly, while feed intake (FI), and gain:feed (G:F) were recorded and calculated on pen basis. Faecal score was evaluated every seven days. On day 0, 9, 18 and 27 blood samples were collected to determine IgA, IgG and haptoglobin concentration. At day 28 all piglets were sacrificed, and tissue samples of ileal Peyer’s patches were collected for the evaluation of IL1α, IL1β, IL6, IL10, TNFα, TLR2, TLR4 and PPARγ gene expression. Nucleotides supplementation significantly increased BW (17.37 vs. 19.00 kg/pig; p =

Early feed restriction of lambs may program animals to achieve reduced feed efficiency traits as a consequence of permanent mitochondrial dysfunction. The hypothesis at the background of the present study is that dietary administration of L-Carnitine (a compound that promotes the activation and transportation of fatty acids into the mitochondria) during the fattening period of early feed restricted lambs can: (a) improve the biochemical profile of early feed restricted lambs, (b) improve feed efficiency, (c) modulate the ruminal and intestinal microbiota, and (d) induce changes in the gastrointestinal mucosa, including the immune status. Twenty-two newborn male Merino lambs were raised under natural conditions but separated from the dams for 9 h daily to allow feed restriction during the suckling period. At weaning, lambs were assigned to a control group being fed ad libitum a complete pelleted diet during the fattening phase (CTRL, n = 11), whereas the second group (CARN, n = 11) received the same diet supplemented with 3 g of L-Carnitine/kg diet. The results revealed that even though L-Carnitine was absorbed, feed efficiency was not modified by dietary L-Carnitine during the fattening period (residual feed intake, p > 0.05), whereas ruminal fermentation was improved [total short-chain fatty acids (SCFAs), 113 vs. 154 mmol/l; p = 0.036]. Moreover, a trend toward increased concentration of butyrate in the ileal content (0.568 vs. 1.194 mmol/100 ml SCFA; p = 0.074) was observed. Other effects, such as reduced heart weight, lower levels of markers related to muscle metabolism or damage, improved renal function, and increased ureagenesis, were detected in the CARN group. Limited changes in the microbiota were also detected. These findings suggest that L-Carnitine may improve ruminal fermentation parameters and maintain both the balance of gut microbiota and the health of the animals. However, the improved ruminal fermentation and the consequent greater accumulation of intramuscular fat might have hidden the effects caused by the ability of dietary L-Carnitine to increase fatty acid oxidation at the mitochondrial level. This would explain the lack of effects of L-Carnitine supplementation on feed efficiency and points toward the need of testing lower doses, probably in the context of animals being fed in excess non-protein nitrogen.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B17y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B17y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report.

There is a growing interest in developing new molecular markers of heart disease in young dogs affected by myxomatous mitral valve disease. The study aimed to measure 3 circulating microRNAs and their application as potential biomarkers in the plasma of Cavalier King Charles Spaniels with early asymptomatic myxomatous mitral valve disease. The hypothesis is that healthy Cavalier King Charles Spaniels have different microRNA expression profiles than affected dogs in American College of Veterinary Internal Medicine (ACVIM) stage B1. The profiles can differ within the same class among subjects of different ages. This is a prospective cross-sectional study. Thirty-three Cavalier King Charles Spaniels in ACVIM stage B1 were divided into three groups (11 younger than 3 years, 11 older than 3 years and younger than 7 years, and 11 older than 7 years), and 11 healthy (ACVIM stage A) dogs of the same breed were included as the control group. Three circulating microRNAs (miR-1-3p, miR30b-5p, and miR-128-3p) were measured by quantitative real-time PCR using TaqMan® probes. Diagnostic performance was evaluated by calculating the area under the receiver operating curve (AUC). MiR-30b-5p was significantly higher in ACVIM B1 dogs than in ACVIM A subjects, and the area under the receiver operating curve was 0.79. According to the age of dogs, the amount of miR-30b-5p was statistically significantly higher in group B17y (2.7 folds, P = 0.018) than in group A. The area under the receiver operating curves were fair in discriminating between group B17y and A (AUC 0.822). Identifying dogs with early asymptomatic myxomatous mitral valve disease through the evaluation of miR-30b-5p represents an intriguing possibility that certainly merits further research. Studies enrolling a larger number of dogs with preclinical stages of myxomatous mitral valve disease are needed to expand further and validate conclusively the preliminary findings from this report.

This manuscript aimed to optimise the encapsulation of Thymus capitatus essential oil into nanoemulsion. Response Surface Methodology results were best fitted into polynomial models with regression coefficient values of more than 0.95. The optimal nanoemulsion showed nanometer-sized droplets (380 nm), a polydispersity index less than 0.5, and a suitable Zeta potential (−10.3 mV). Stability results showed that nanoemulsions stored at 4 °C were stable with the lowest d3,2, PolyDispersity Index (PDI), and pH (day 11). Significant ameliorations in the capacity to neutralise DPPH radical after the encapsulation of the antimicrobial efficacy of thyme essential oil were recorded. S. typhimurium growth inhibition generated by nanoencapsulated thyme essential oil was 17 times higher than by bulk essential oil. The sensory analysis highlighted that the encapsulation of thyme essential oil improved enriched milk’s sensory appreciation. Indeed, 20% of the total population attributed a score of 4 and 5 on the scale used for milk enriched with nanoemulsion. In comparison, only 11% attributed the same score to milk enriched with bulk essential oil. The novel nanometric delivery system presents significant interest for agroalimentary industries.

L’Educazione fisica, le attività motorie e lo sport, nelle Università italiane, sono ambiti disciplinari presenti nei corsi triennali di laurea in Scienze delle attività motorie e sportive e nei corsi biennali di laurea magistrali, successivi al percorso formativo degli ex Istituti Superiori di Educazione Fisica (I.S.E.F.). I piani di studio dei corsi di laurea prevedono una struttura multi-inter-disciplinare in cui sono presenti, in particolare, due settori scientifico-didattici, definiti di base e caratterizzanti, denominati metodi e didattiche delle attività motorie e metodi e didattiche delle attività sportive. Nell’ambito di ciascun settore sono compresi diversi insegnamenti ciascuno con una propria struttura disciplinare, valenze formative e vincoli epistemologici, peculiari metodologie di ricerca, caratterizzanti il profilo professionale dello studente. Gli obiettivi del seguente contributo riguardano una duplice prospettiva: analizzare la struttura dei settori disciplinari Metodi e Didattiche delle attività motorie e Metodi e Didattiche delle attività sportive e dei relativi contenuti essenziali e caratterizzanti; orientare la definizione del Syllabus necessario a definire chiaramente, per ciascun insegnamento, obiettivi e competenze attese, contenuti e metodi didattici, durata e modalità di valutazione, con particolare riferimento al settore concorsuale denominato Didattica, Pedagogia Speciale e Ricerca educativa (11D/2), al fine di condividere con gli studenti e gli stakeholder il processo didattico ed il profilo professionale in uscita. Si presentano qui i contenuti essenziali dell’insegnamento di Teoria e Metodologia del Movimento Umano, compreso nel ssd M-EDF/01- Metodi e Didattiche delle Attività Motorie. I rapporti tra settori scientifico-disciplinari e settori concorsuali, in Italia, costituiscono un snodo essenziale ed ineludibile per superare ogni possibile antitesi scientifica e metodologica, per condividere e definire i rapporti tra i temi della didattica e della ricerca, le strategie per promuovere l’evoluzione delle conoscenze e delle competenze in uscita dello Studente. Il contributo scaturisce dal bisogno di definire e condividere il core curriculum dei suddetti settori scientifico-disciplinari in ambito didattico e pedagogico, individuando altresì relazioni interdisciplinari e metodologie della ricerca.

Numerosi studi nell’ambito dell’educazione dell’infanzia attestano che il bambino è attivo protagonista del proprio sviluppo e, nell’interazione con l’ambiente, conosce sé stesso, sperimenta la gioia di vivere e la responsabilità di prendersi cura di ciò che lo circonda. Il presente contributo, senza alcuna pretesa di esaustività, pone a tema il valore della cura di sé muovendo, in una prospettiva ecologica e sostenibile, dall’ascolto del sé interiore e del lin-guaggio artistico-musicale per arrivare alla scoperta dell’alterità. In tale orizzonte interpretativo, a chi educa è richiesto di aver cura di sé per progettare ed offrire, secondo una logica riflessivo - trasformativa, ambienti, esperienze e percorsi educativi sostenibili che consentano al bambino di po-tenziare il proprio sé e di dare al contempo un significativo apporto alla co-struzione nella ricerca del bene comune.

MicroRNAs (miRNAs) are small and highly conserved non-coding RNA molecules that orchestrate a wide range of biological processes through the post-transcriptional regulation of gene expression. An intriguing aspect in identifying these molecules as biomarkers is derived from their role in cell-to-cell communication, their active secretion from cells into the extracellular environment, their high stability in body fluids, and their ease of collection. All these features confer on miRNAs the potential to become a non-invasive tool to score animal welfare. There is growing interest in the importance of miRNAs as biomarkers for assessing the welfare of livestock during metabolic, environmental, and management stress, particularly in ruminants, pigs, and poultry. This review provides an overview of the current knowledge regarding the potential use of tissue and/or circulating miRNAs as biomarkers for the assessment of the health and welfare status in these livestock species.

The great adaptability, productivity and importance in human diet conditioned the diffusion of potato (Solanum tuberosum L.) in very different areas, leading to the formation of numerous landraces through anthropic or natural selection. Genoa mountainous inland is an historical area for potato cultivation and landraces are preserved by farmers associations as “Consorzio della Quarantina”. The aim of this study was the phytochemical and morphometric characterization of three potato landraces of the consortium (Quarantina Bianca, Quarantina Prugnona and Rubra spes) analysing the bio-agronomical performance at different elevations. The commercial cultivar Kennebec was used as control. For the morphometric analysis, the results of pairwise MANOVA shows that only Quarantina Bianca does not present significant differences with Kennebec in the mean shape. The four potato varieties resulted significantly different for the most of the considered phytochemical aspects (among content in ash, starch, solanine, total flavonoids, total phenols, chlorogenic acid, caffeic acid, ferulic acid and radical scavenging activity only starch and solanine content didn’t result statistically different). A remarkable result was a more consistent yield for all the varieties with the increase of elevation in the agronomical trials. We can conclude that Genovese landraces are a good choice to exploit Genovese mountain marginal territories.

Otitis externa is one of the most common diseases in dogs. It is associated with bacteria and yeast, which are regarded as secondary causes. Cerumen is a biological substance playing an important role in the protection of ear skin. The involvement of cerumen in immune defense is poorly understood. MicroRNAs can modulate the host immune response and can provide promising biomarkers for several inflammatory and infectious disorder diagnosis. The aims of this study were to profile the cerumen miRNA signature associated with otitis externa in dogs, integrate miRNAs to their target genes related to immune functions, and investigate their potential use as biomarkers. Cerumen was collected from healthy and otitis affected dogs and the expression of miRNAs was profiled by Next Generation Sequencing; the validation of the altered miRNAs was performed using RT-qPCR. The potential ability of miRNAs to modulate immune-related genes was investigated using bioinformatics tools. The results pointed out that 32 miRNAs, of which 14 were up- and 18 down-regulated, were differentially expressed in healthy vs. otitis-affected dogs. These results were verified by RT-qPCR. To assess the diagnostic value of miRNAs, ROC analysis was carried out, highlighting that 4 miRNAs are potential biomarkers to discriminate otitis-affected dogs. Bioinformatics showed that cerumen miRNAs may be involved in the modulation of host immune response. In conclusion, we have demonstrated for the first time that miRNAs can be efficiently extracted and quantified from cerumen, that their profile changes between healthy and otitis affected dogs, and that they may serve as potential biomarkers. Further studies are necessary to confirm their diagnostic value and to investigate their interaction with immune-related genes.

Once a very important crop, the cultivation of hemp has seen a significant reduction in Europe for a long time, but it is now subject to direct payments and promotion initiatives. Italy used to be an important producer and exporter of hemp textiles until the Second World War but currently information is lacking regarding the hemp production chain and legislation on the issue is often misunderstood by producers. Moreover, there has been an important development of economic activities connected with hemp, such as the so-called “hemp shops” or “grow shops” and the market of a product called “cannabis light”, the dried inflorescences of industrial hemp. The aim of the study was to investigate 30 Italian hemp farms in order to identify the characteristics of the production chain and the uses of the crop. Some considerations on this sector in the Italian economic and legislative contest are made and an anonymous web survey on the commercial activities associated with hemp (grow shops) was conducted. Most farms are multifunctional, 83% have been set up recently (in the last ten years) and directly as hemp producers. They are run by young entrepreneurs (57% holders under 35) and allow the use of marginal abandoned territories (43% of the farms). The 30 farms cultivate 460 ha of hemp and the extension of the field crops is very variable, from small patches in the mountains of 0,001 ha to more than 100 ha farms in the plains (in particular in Campania region). Almost all the farms use the crop to produce more than one end-product (seeds, flour, decorticated seeds, hemp-beer, seeds for animal nutrition and food oil from seeds, seeds and inflorescences harvested for cosmetic use, herbal use and extraction of active ingredients). In some cases, “technical use” linked to selling of the dry top inflorescences of industrial hemp for smoking was declared and it was found that there has been a significant increase in grow shops in Italy, from 4 in 2002 to more than 700 in 2018. As emerges from the analyses of European and Italian legislation, there is a need for clear regulations and a system of control by regulatory organizations considering the actual criticalities. At the same time, the renewed appeal of this crop derives from the multiple possibilities of use of the plant and from growing consumer demand for eco-compatible and sustainable products.

The aim of this study was to examine the reference range of blood plasma fibrinogen, which belongs to the group of moderate acute phase protein in rabbits. The experiment followed the dynamics of blood plasma fibrinogen (Fb) during the first year of life in 12 New Zealand White rabbits, 6 males and 6 females, starting at 1 month of age until the 7th month. It was shown that fibrinogen concentra-tions increased with the age. There was a statistically significant difference in Fb concentrations in rabbits at 3 (P

The phenomenological pedagogy has always been focused its attention to the centrality of the subject, on his concrete experience and on the related embodied aspects referred to the consciousness and awareness of the body and action. According with the Embodied Cognition theory, phenomenology is outlining a pedagogical framework defined as Embodied Education. Within this pedagogical approach, physical education and sport sciences are enhancing the educational strategy based on the involvement of the subject as a fundamental actor of his educational processes. This contribution aims to underline the intrinsically embodied essence of motor activity, game and sport, and the educational strategies that enhance the body as a subject that creates cognition through the interaction with the environment-objects- persons, in the concrete situation experienced by the subject.

The aim of the present study was to determine plasma haptoglobin concentrations in cows in field conditions. Experiments were carried out a total of 106 cows, divided in 5 groups. The 1st group comprised 25 cows with low milk somatic cell counts (SCC), without microbiological findings used as controls; the IInd group –23 cows with untreated subclinical mastitis with high milk SCC and with-out microbiological findings; the IIIrd group: 13 cows with untreated subclinical mastitis with high milk SCC and with microbiological findings. The IVth group consisted of 17 cows with subclinical mastitis, high milk SCC, microbiological findings, treated with tetracycline HCL 200 mg, neomycin 250 mg, bacitracin 2000 IU, prednisolone 10 mg (Mastijet fort®, MSD Animal Health), and the Vth group – 28 cows with clinical mastitis treated with Mastijet fort®, MSD Animal Health. Our results showed that the mean plasma haptoglobin concentrations in the I (control) group was 0.054±0.003 g/L. In the IInd group (0.288±0.04 g/L) Hp was 5.2 times higher that the mean values of controls (P

Circulating microRNAs (miRNAs) are emerging as promising biomarkers for several disorders and related pain. In equine practice, acute laminitis is a common disease characterised by intense pain that severely compromises horse welfare. Recently, the Horse Grimace Scale (HGS), a facial expression-based pain coding system, was shown to be a valid welfare indicator to identify pain linked to acute laminitis. The present study aimed to: determine whether miRNAs can be used as biomarkers for acute pain in horses (Equus caballus) affected by laminitis; integrate miRNAs to their target genes and to categorise target genes for biological processes; gather additional evidence on concurrent validity of HGS by investigating how it correlates to miRNAs. Nine horses presenting acute laminitis with no prior treatment were recruited. As control group, nine healthy horses were further included in the experimental design. Samples were collected from horses with laminitis at admission before any treatment (‘pre-treatment’) and 7 days after routine laminitis treatment (‘post-treatment’). The expression levels of nine circulating miRNAs, namely hsa-miR-532-3p, hsa-miR-219-5p, mmu-miR-134-5p, mmu-miR-124a-3p, hsa-miR-200b-3p, hsa-miR-146a-5p, hsa-miR-23b-3p, hsa-miR-145-5p and hsa-miR-181a-5p, were detected and assessed as potential biomarkers of pain by quantitative PCR using TaqMan® probes. The area under the receiver operating curve (AUC) was then used to evaluate the diagnostic performance of miRNAs. Molecular data were integrated with HGS scores assessed by one trained treatment and time point blind veterinarian. The comparative analysis demonstrated that the levels of miR-23b-3p (P=0.029), miR-145-5p (P=0.015) and miR-200b-3p (P=0.023) were significantly higher in pre-treatment and the AUCs were 0.854, 0.859 and 0.841, respectively. MiR-200b-3p decreased after routine laminitis treatment (P=0.043). Combining two miRNAs in a panel, namely miR-145-5p and miR-200b-3p, increased efficiency in distinguishing animals with acute pain from controls. In addition, deregulated miRNAs were positively correlated to HGS scores. Computational target prediction and functional enrichment identified common biological pathways between different miRNAs. In particular, the glutamatergic pathway was affected by all three miRNAs, suggesting a crucial role in the pathogenesis of pain. In conclusion, the dynamic expression of circulating miR-23b-3p, miR-145-5p and miR-200b-3p was detected in horses with acute laminitis and miRNAs can be considered potentially promising pain biomarkers. Further studies are needed in order to assess their relevancy in other painful conditions severely compromising horse welfare. An important implication would be the possibility to use them for the concurrent validation of non-invasive indicators of pain in horses.

Most acute intestinal diseases are caused by food-borne pathogens. A fast and simple real-time PCR-based procedure for simultaneous detection of food contamination by any of the five food-borne pathogens: Campylobacter jejuni, Mycobacterium bovis, Enterobacter sakazaki, Shigella boydii, Clostridium perfrigens using multiplex EvaGreen real-time PCR for LightCycler was developed and evaluated. Real-time qPCR showed excellent sensitivity. Tm calling and Melting Curve Genotyping (MCG) were used for analysis of PCR product melting curves. The Melting Curve Genotyping option showed good performance for discrimination of positive samples containing DNA of single pathogen or pathogen mixtures from negative samples.

Otitis externa is a common multifactorial disease in dogs, characterized by broad and complex modifications of the ear microbiota. The goal of our study was to describe the ear cerumen microbiota of healthy dogs, within the same animal and between different animals, and to compare the cerumen microbiota of otitis affected dogs with that of healthy animals. The present study included 26 healthy dogs, 16 animals affected by bilateral otitis externa and 4 animals affected by monolateral otitis externa. For each animal cerumen samples from the right and left ear were separately collected with sterile swabs, and processed for DNA extraction and PCR amplification of the 16S rRNA gene. Amplicon libraries were sequenced using an Ion Torrent Personal Genome Machine (PGM), and taxonomical assignment and clustering were performed using QIIME 2 software. Our results indicate that the bacterial community of the cerumen in healthy dogs was characterized by extensive variability, with the most abundant phyla represented by Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes and Fusobacteria. The analysis of both alpha and beta diversity between pairs of left and right ear samples from the same dog within the group of affected animals displayed higher differences than between paired samples across healthy dogs. Moreover we observed reduced bacterial richness in the affected group as compared with controls and increased variability in population structure within otitis affected animals, often associated with the proliferation of a single bacterial taxon over the others. Moreover, Staphylococcus and Pseudomonas resulted to be the bacterial genera responsible for most distances between the two groups, in association with differences in the bacterial community structure. The cerumen microbiota in healthy dogs exhibits a complex bacterial population which undergoes significant modifications in otitis affected animals.

Saffron (Crocus sativus L.) is the most expensive spice in the world and is used in food, cosmetic and dyeing industries. Considering that the production of saffron is increasingly widespread in medium-small Italian farms as well as the scarceness of information and studies regarding the quality of the saffron produced in Italy, the principal aim of this study was to investigate the quality of Italian saffron. Qualitative analysis was conducted in accordance with ISO 3632 1,2:2010-2011 considering 484 samples collected over four years (2015-2018). In particular, moisture content, aroma strength (safranal), colouring strength (crocin) and flavour strength (picrocrocin) were assessed for each sample, and whether spice quality varied according to the geographical area where the spice was produced was also investigated. Qualitative analysis showed that the majority (84-93%) of the samples analysed are of the first quality category, regardless of the year of production. Moisture content and colouring strength are the factors that influence the quality of the spice most. Principal component analysis showed that quality is not influenced by the geographical area where the spice was produced. Finally, some best agricultural practices to obtain a high quality saffron spice are reported.

Fare scuola oggi richiede una didattica innovativa che coinvolga spazi, tempi e attività diversificate tali da spostare l’attenzione dall’aula, come unico ambiente di apprendimento, per aprirsi a una molteplicità di ambienti capaci di coinvolgere in forma attiva e personalizzata ogni singolo allievo. Le linee guida ministeriali forniscono una idea chiara sulla strada da intraprendere per dare vita a una scuola innovativa. L’analisi parziale di un questionario, proposto a un campione di 631 allievi di scuola primaria e secondaria di primo grado, sembra indicare come i tempi siano maturi per iniziare tale percorso innovativo. Gli attuali studenti, probabilmente sollecitati da ambienti extrascolastici innovativi e in continua evoluzione, manifestano bisogni e attese in piena sintonia con l’idea di una scuola nuova, dinamica, attiva e pienamente coinvolgente. Una scuola in cui gli aspetti corporei, motori ed emotivi, non possono più essere segregati tra banco e sedia, ma devono aprirsi a una varietà di situazioni educative cui non si può più rinunciare.

Using plant essential oils (EOs) contributes to the growing number of natural plants’ applications in livestock. Scientific data supporting the efficacy of EOs as anti-inflammatory, antibacterial and antioxidant molecules accumulates over time; however, the cumulative evidence is not always sufficient. EOs antioxidant properties have been investigated mainly from human perspectives. Still, so far, our review is the first to combine the beneficial supporting properties of EOs in a One Health approach and as an animal product quality enhancer, opening new possibilities for their utilization in the livestock and nutrition sectors. We aim to compile the currently available data on the main anti-inflammatory effects of EOs, whether encapsulated or not, with a focus on mammary gland inflammation. We will also review the EOs’ antioxidant activities when given in the diet or as a food preservative to counteract oxidative stress. We emphasize EOs’ in vitro and in vivo ruminal microbiota and mechanisms of action to promote animal health and performance. Given the concept of DOHaD (Developmental Origin of Health and Diseases), supplementing animals with EOs in early life opens new perspectives in the nutrition sector. However, effective evaluation of the significant safety components is required before extending their use to livestock and veterinary medicine.

Water buffalo mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies to avoid or reduce antibiotics' based therapies, in particular during subclinical mastitis. The use of Generally Regarded As Safe bacteria (GRAS) such as Lactobacillus rhamnosus to restore the unbalance in mammary gland microbiota could provide potential corrective measures. The aim of this study was to investigate the changes in milk microbiota after the intramammary treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters naturally affected by subclinical mastitis as compared to antibiotic therapy.A number of 43 quarters affected by subclinical mastitis with no signs of clinical inflammation and aerobic culture positive for pathogens were included in the study. The experimental design was as follows: 11 quarters were treated with antibiotics, 15 with inactivated cultures of Lactobacillus rhmnosus and 17 with PBS as negative control, by means of intrammary injection. Samples were collected at eight time points, pre- (T-29, T-21, T-15, T-7, T0 days) and post- treatment (T1, T2, and T6 days). Microbiological culture and Somatic Cell Count (SCC) were perfomed on all the samples, and microbiota was determined on milk samples collected at T0 and T6 by amplifying the V4 region of 16S rRNA gene by PCR and sequencing using next generation sequencing technique. Treatment with Lactobacillus rhamnosus elicited a strong chemotactic response, as determined by a significant increase of leukocytes in milk, but did not change the microbiological culture results of the treated quarters. For what concerns the analysis of the microbiota, the treatment with Lactobacillus rhamnosus induced the modification in relative abundance of some genera such as Pseudomonas and 5-7N15. As expected, antibiotic treatment caused major changes in microbiota structure with an increase of Methylobacterium relative abundance. No changes were detected after PBS treatment. In conclusion, the present findings demonstrated that the in vivo intrammmary treatment with Lactobacillus rhamnosus has a transient pro-inflammatory activity by increasing SCC and is capable to modify the microbiota of milk after six days from inoculation, albeit slightly, even when the bacterial cultures were heat inactivated. Further studies are necessary to assess the potential use of this GRAS as supportive therapy against mastitis.

L’educazione motoria-fisica-sportiva si sta caratterizzando sempre di più, grazie agli apporti delle scienze pedagogiche, per una didattica qualitativa rispondente ai bisogni e alle necessità dei bambini. A partire dall’ambiente scolastico, luogo elettivo di formazione dei futuri cittadini, l’idea di una educazione attraverso il corpo e movimento, con caratteristiche di piacere, divertimento, coinvolgimento cognitivo ed emotivo, sta influenzando anche gli ambienti sportivi. Il presente contributo, condividendo l’importanza e la necessità di questo approccio qualitativo, dell’educazione motoria-fisica-sportiva, solleva il problema riferito alla necessità di porre attenzione anche all’approccio quantitativo in risposta alle carenze che le attuali generazioni infantili stanno evidenziando a livello psico-fisico e, in particolare, nella funzionalità aerobica. Dopo aver presentato uno screening, su un campione di bambini/e di undici anni, in ingresso nella scuola secondaria di primo grado, a conferma delle carenze appena richiamate, si definiscono alcune possibili strategie integrate, qualitative e quantitative, in grado di rispondere alle emergenti problematichedi benessereche riguardano l’età evolutiva.

MicroRNA (miRNA) have been identified in circulating blood and might have the potential to be used as biomarkers for several pathophysiological conditions. To identify miRNA that are altered following stress events, turkeys (Meleagris gallopavo) were subjected to 2 h of road transportation. The expression levels of five circulating miRNA, namely miR-22, miR-155-5p, miR-181a-3p, miR-204 and miR-365-3p, were detected and assessed by quantitative polymerase chain reaction using TaqManR probes, as potential biomarkers of stress. The areas under the receiver operating characteristic curves were then used to evaluate the diagnostic performance of miRNA. A panel of three stress-responsive miRNA, miR-22, miR-155 and miR-365 were identified; their expression levels were significantly higher after road transportation and the area under the curve (AUC) were 0.763, 0.71 and 0.704, respectively. Combining the three miRNA a specificity similar to the one found for the three miRNA separately was found. The AUC of the weighted average of the three miRNA was 0.763. This preliminary study suggests that the expression levels of circulating miR-22, miR-155 and miR-365 are increased during transport-related stress and that they may have diagnostic value to discriminate between stressed- and unstressed animals.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid – i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002 – Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East–West and North–South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.