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This research delves into the impact of substitution of 7% of poultry meal (PM) with hydrolyzed feather meal (HFM) on the animal performance and fecal microbiota of dogs. The study was conducted on six adult female English setter dogs, split into control (CTR), and treated (TRT) dietary treatment groups, with similarity for age, initial body weight, and body condition score. During 45-days, body weight, body condition score, muscle condition score, fecal consistency score and microbiota were monitored at the beginning of the trial and after 3, 7, 15 and 45 days. No significant differences (P > 0.05) were observed for these parameters over the whole period of the trial, neither in relation to diet, nor to time, nor to the interaction diet x time. Significant differences were observed for alpha between diets and the TRT group displayed higher evenness compared to the CTR group. Beta diversity analysis revealed distinctions in microbiota composition between the CTR and TRT groups, with the former demonstrating higher biodiversity. Discriminant analysis highlighted 9 significant taxa and 6 of them were significantly different at the Kruskal Wallis test between diets. The results showed an increase in relative abundance (RA) for the CTR group of Streptococcus, Colinsella stercoris, Ruminococcus gnavus, and Bacteroides coprophilus. Conversely, higher RA was observed in the TRT group for Peptostreptococcaceae and Bacteroides uniformis. These findings indicated that the inclusion of hydrolyzed feather meal in the diet of dogs is well accepted and do not have adverse effects in the parameters analyzed., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study aimed to determine the impact of a novel formulation of a supplement composed of the natural ingredients, bromelain, quercetin, and Lentinula edodes , on the gut microbiota of healthy adult dogs. Adult healthy female dogs were administered either a placebo (CTR, n = 15) or the supplement (TRT, n = 15) over 28 days. Stool samples were collected for 16S rRNA sequencing before supplement administration (T0), at completion of supplement administration (T28), and one week after the end of supplement administration (T35) to characterize changes in the gut microbial communities. QIIME was used to determine both alpha- and beta-diversity, and ANCOM-BC was used to identify differences in taxonomic abundances before and after supplementation. We found a significant decrease in overall diversity in the CTR group but no significant differences in overall diversity in the TRT group over time. Furthermore, we found differences in the abundance of several taxa in both the CTR and TRT groups, but differences in the abundance of beneficial bacteria were more pronounced in the TRT group. Specifically, we found increases in the abundance of sequences belonging to the genera Bifidobacterium , Lactobacillus , and Pediococcus at T28 in the TRT group with significant increases in Bifidobacterium and Lactobacillus persisting at T35 when compared to T0. Importantly, members of these genera are considered important for their anti-inflammatory properties, vital for fostering a balanced and robust gut microbiota in dogs. The results of our study show the potential of our supplement to selectively enhance specific beneficial bacterial taxa, offering a targeted approach to modulating the gut microbiome without causing disruptions to the overall equilibrium., Competing Interests: The authors declare no conflicts of interest.

Overweight and obese dogs can develop metabolic dysfunction, characterized by an inflammatory response and involvement of liver functions. If a modulation of the gut microbiome and its interaction with the gut-liver axis is implicated in the development of metabolic dysfunction, exploration becomes necessary. Over the past decade, diverse therapeutic approaches have emerged to target pathogenic factors involved in metabolic dysfunction. This study investigated the impact of a supplement with hepatoprotective activity, containing extracts of Silybum marianum , prebiotics, probiotics, n-3 polyunsaturated fatty acids, vitamins, and minerals on hematological markers of liver functions and inflammation, as well as on the intestinal microbiota of 10 overweight adult dogs over a 35-day time span. Animals underwent clinical and laboratory evaluations every 7 days, both before the administration of the supplement (T0) and after 7, 14, 21, 28, and 35 days (T1, T2, T3, T4, and T5). In comparison to T0, a significant ( p < 0.05) decrease in ALP, glucose, direct bilirubin, and CRP was observed from T3 to T5. The alpha diversity of the fecal microbiota significantly decreased ( p < 0.05) only at T1, with high variability observed between dogs. Total short-chain fatty acid and lactic acid were also lower at T1 ( p < 0.05) compared to the other times of sampling. The beta diversity of the fecal microbiota failed to show a clear pattern in relation to the sampling times. These results of blood parameters in overweight dogs show a reduction of the inflammation and an improvement of metabolic status during the study period, but the effective contribution of the supplement in this clinical outcome deserves further investigation. Furthermore, the considerable individual variability observed in the microbiome hinders the confident detection of supplement effects.

Costs of production have deeply increased each year in the last decades, breeders are continuously looking for more cost effective and more efficient ways to produce milk. Despite the major signs of progress in productivity, it is fundamental to optimize rather than maximize the performances of the dairy cows. Mastitis is still a highly prevalent disease in the dairy sector which causes several economic losses and environmental effect. Its accurate and early diagnosis is crucial to improve profitability of dairy cows and contribute to a more sustainable dairy industry. Among mastitis reduction strategies, there is the urgent need to implement breeding objectives to select cows displaying mastitis resistance by investigating the genetic mechanisms at the base of the inflammatory response. Therefore, in this study we aimed to further understand the genetic background of the differential somatic cell count (DSCC), which provides thorough insights on the actual inflammatory status of the mammary glands. The objectives of this study were to estimate on a cohort of 20,215 Italian Simmental cows over a 3-yr period: (1) the heritability and repeatability values of somatic cell score (SCS) and DSCC, (2) the genetic and phenotypic correlations between these 2 traits and milk production and milk composition traits, (3) the heritability and repeatability values of SCS and DSCC within class of udder health status. Heritability was low both for SCS (0.06) and DSCC (0.08), whereas the repeatability values for these traits were 0.43 and 0.36, suggesting that the magnitude of cow permanent environmental effect for these traits is remarkable. The genetic and phenotypic correlation of SCS with DSCC was 0.612 and 0.605, respectively. Because both significantly differed from the unit, we must consider those traits as different ones. This latter aspect corroborates the need to consider the DSCC as a further indicator of inflammatory status which might be implemented in the Simmental breed genetic evaluation. It is worthy to mention that heritability estimates for SCS and DSCC were the highest in healthy cows compared with the other udder health classes. This implies that when the udder health status changes, it is most likely due to environmental factors rather than aspects related to the animal's genetics. In contrast, the highest additive genetic variance and heritability found for SCS and DSCC in the healthy group might reveal the potential to further implement breeding strategies to select for healthier animals., (© 2023, The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Oxidative stress causes several pathological conditions in humans and animals, including gastrointestinal disorders. The aim of this study was to analyze the antioxidant capacity of three natural powdered raw materials containing quercetin, bromelain, and Lentinula edodes and develop a new feed supplement for dogs using a combination of them. The total phenolic content (TPC), antioxidant activity, DPPH (2,2-diphenyl-1-picrylhydrazyl), and ABTS (2,2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) of the extracts, either individually or in combination, were evaluated colorimetrically. The effects of this supplement on healthy adult dogs' nutritional, inflammatory, and stress status were evaluated. American Staffordshire Terrier adult female dogs ( n = 30) were randomly assigned to a control ( n = 15) or a treated ( n = 15) group. The supplement was added as powder to the food of the treated dogs once daily for 28 days. There was no significant difference in the body weight and body condition scores between the initial and final phases of the experiment. At the end of our study, a significant decrease in fecal calprotectin, cortisol, indole/skatole, and N-methylhistamine and a significant increase in short-chain fatty acids were observed as compared to the control group. In conclusion, this natural feed supplement can be used to improve gastrointestinal health and psycho-physical conditions in dogs.

Recent studies have found bacterial DNA in the blood of healthy individuals. To date, most studies on the blood microbiome have focused on human health, but this topic is an expanding research area in animal health as well. This study aims to characterize the blood microbiome of both healthy dogs and those with chronic gastro-enteropathies. For this study, blood and fecal samples were collected from 18 healthy and 19 sick subjects, DNA was extracted through commercial kits, and the V3-V4 regions of the 16S rRNA gene were sequenced on the Illumina platform. The sequences were analyzed for taxonomic annotation and statistical analysis. Alpha and beta diversities of fecal microbiome were significantly different between the two groups of dogs. Principal coordinates analysis revealed that healthy and sick subjects were significantly clustered for both blood and fecal microbiome samples. Moreover, bacterial translocation from the gut to the bloodstream has been suggested because of found shared taxa. Further studies are needed to determine the origin of the blood microbiome and the bacteria viability. The characterization of a blood core microbiome in healthy dogs has potential for use as a diagnostic tool to monitor for the development of gastro-intestinal disease.

The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau&#95;5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows.

The review described the most important factors affecting the development of the intestinal microbiota in puppies from birth to weaning. The health and well-being of the microbiome in puppies is influenced by the type of parturition, the maternal microbiota, and the diet of the mother, directly or indirectly. The isolation of bacteria in dogs from the placenta, fetal fluids, and fetuses suggests that colonization could occur before birth, although this is still a matter of debate. Accordingly, newborn puppies could harbor bacteria that could be of maternal origin and that could influence microbial colonization later in life. However, the long-term impacts on health and the clinical significance of this transfer is not yet clear and needs to be investigated. The same maternal bacteria were found in puppies that were born vaginally and in those delivered via cesarean section. Potentially, the relationship between the type of parturition and the colonization of the microbiome will influence the occurrence of diseases, since it can modulate the gut microbiome during early life. In addition, puppies' gut microbiota becomes progressively more similar to adult dogs at weaning, as a consequence of the transition from milk to solid food that works together with behavioral factors. A number of researches have investigated the effects of diet on the gut microbiota of dogs, revealing that dietary interference may affect the microbial composition and activity through the production of short-chain fatty acids and vitamins. These compounds play a fundamental role during the development of the fetus and the initial growth of the puppy. The composition of the diet fed during pregnancy to the bitches is also an important factor to consider for the health of newborns. As far as it is known, the effects of the type of parturition, the maternal microbiota, and the diet on the microbial colonization and the long-term health of the dogs deserve further studies. Definitely, longitudinal studies with a larger number of dogs will be required to assess a causal link between microbiome composition in puppies and diseases in adult dogs.

The aim of this study was to quantify some environmental (individual herds, herd productivity, milking system, and season) and animal factors [individual animals, breed, days in milk (DIM) and parity] on the variability of the log-10 transformation of somatic cell count (LSCC) and differential somatic cell count (DSCC) on individual bovine milk. A total of 159,360 test-day records related to milk production and composition were extracted from 12,849 Holstein-Friesian and 9,275 Simmental cows distributed across 223 herds. Herds were classified into high and low productivity, defined according to the average daily milk net energy output (DMEO) yielded by the cows. Data included daily milk yield (DYM; kg/d), milk fat, protein, lactose, SCC, and DSCC, and information on herds (i.e., productivity, milking system). The daily production of total and differential somatic cells in milk was calculated and then log-10 transformed, obtaining DLSCC and DLDSCC, respectively. Data were analyzed using a mixed model including the effects of individual herd, animal, repeated measurements intra animal as random, and herd productivity, milking system, season, breed, DIM, parity, DIM × parity, breed × season, DIM × milking system and parity × milking system as fixed factors. Herds with a high DMEO were characterized by a lower content of LSCC and DSCC, and higher DLSCC and DLDSCC, compared to the low DMEO herds. The association between milking system and somatic cell traits suggested that the use of the automatic milking systems would not allow for a rapid intervention on the cow, as evidenced by the higher content of all somatic cell traits compared to the other milking systems. Season was an important source of variation, as evidenced by high LSCC and DSCC content in milk during summer. Breed of cow had a large influence, with Holstein-Friesian having greater LSCC, DSCC, DLSCC, and DLDSCC compared to Simmental. With regard to DIM, the variability of LSCC was mostly related to that of DSCC, showing an increase from calving to the end of lactation, and suggesting the higher occurrence of chronic mastitis in cows toward the end of lactation. All the somatic cell traits increased across number of parities, possibly because older cows may have increased susceptibility to intramammary infections., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The aim of this study was to investigate the effects on gene expression in canine fibroblasts after incubation with a medium enriched with atopic dermatitis canine serum (CAD) compared with healthy canine serum (CTRL) and fetal bovine serum (FBS). Differential Expression and Pathway analysis (iDEP94) in R package (v0.92) was used to identify differentially expressed genes (DEGs) with a False Discovery Rate of 0.01. DEGs from fibroblasts incubated with CAD serum were significantly upregulated and enriched in the extracellular matrix (ECM) and focal adhesion signalling but downregulated in the oxidative phosphorylation pathway. Genes involved in profibrotic processes, such as TGFB1, INHBA, ERK1/2, and the downward regulated genes (collagens and integrins), were significantly upregulated after fibroblasts were exposed to CAD serum. The observed downregulation of genes involved in oxidative phosphorylation suggests metabolic dysregulation toward a myofibroblast phenotype responsible for fibrosis. No differences were found when comparing CTRL with FBS. The DEGs identified in fibroblasts incubated with CAD serum suggest activation of signalling pathways involved in gradual differentiation through a myofibroblast precursors that represent the onset of fibrosis. Molecular and metabolic knowledge of fibroblast changes can be used to identify biomarkers of the disease and new potential pharmacological targets., (© 2022. The Author(s).)

The aim of this study was to evaluate the effect of the administration of Saccharomyces boulardii on the nutritional, immunological, inflammatory, and stress status and on the composition of the gut microbiota and mycobiota in healthy adult dogs. A total of 25 American Staffordshire Terrier dogs were selected and randomly assigned to two groups: control (CTR, n = 12) and treated (TRT, n = 13) groups. No significant differences were found between the two groups regarding body weight, body condition score, and fecal score. No significant differences in microbiota/mycobiota, short chain fatty acids, indole/skatole, histamine, zonulin, or lactoferrin were detected. Indeed, supplementation with S. boulardii significantly decreased fecal calprotectin Immunoglobulin A, indicating an improvement in the gut well-being. Interestingly, fecal cortisol significantly decreased in dogs belonging to the TRT group compared to the CTR, suggesting both an improvement of the intestinal status and a reduction of stress, a common condition affecting animals managed in a breeding environment.

Protected Designation of Origin (PDO) dry-cured ham is the most important product in the Italian pig breeding industry, mainly oriented to produce heavy pig carcasses to obtain hams of the right weight and maturity. Recently, along with the traditional traits swine breeding programs have aimed to include novel carcass traits. The identification at the genome level of quantitative trait loci (QTLs) affecting such new traits helps to reveal their genetic determinism and may provide information to be integrated in prediction models in order to improve prediction accuracy as well as to identify candidate genes underlying such traits. This study aimed to estimate genetic parameters and perform a single step genome wide association studies (ssGWAS) on novel carcass traits such as untrimmed (UTW) and trimmed thigh weight (TTW) in two pig crossbred lines approved for the ham production of the Italian PDO. With this purpose, phenotypes were collected from ~1800 animals and 240 pigs were genotyped with Illumina PorcineSNP60 Beadchip. The single-step genomic BLUP procedure was used for the heritability estimation and to implement the ssGWAS. QTL were characterized based on the variance of 10-SNP sliding window genomic estimated breeding values. Moderate heritabilities were detected and QTL signals were identified on chromosome 1, 4, 6, 7, 11 and 15 for both traits. As expected, the genetic correlation among the two traits was very high (~0.99). The QTL regions encompassed a total of 249 unique candidate genes, some of which were already reported in association with growth, carcass or ham weight traits in pigs. Although independent studies are required to further verify our findings and disentangle the possible effects of specific linkage disequilibrium in our population, our results support the potential use of such new QTL information in future breeding programs to improve the reliability of genomic prediction.

Mastitis is an inflammatory disease of the mammary gland, caused by the invasion of microorganism on this site, associated with an altered immune response. Recent studies in this field hypothesize that the origin of these pathogens can also be from the gastrointestinal tract, through the entero-mammary pathway in relation to an increase in gut permeability. In this study, we wanted to investigate if inflammatory status of the mammary gland is related to an alteration of gut permeability. The microbiome of feces, blood and milk of lactating cows, recruited on the basis of the total somatic cell count and of the percentage of polymorphonuclear neutrophils and lymphocytes, was studied. Cows were divided into healthy (G), at risk of mastitis (Y) and with mastitis (R) classifications. The bacterial DNA was extracted and the V3 and V4 regions of 16S rRNA sequenced. Moreover, the quantification of total bacteria was performed with quantitative real-time PCR. A non-parametric Kruskal-Wallis test was applied at the phylum, family and genera levels and beta biodiversity was evaluated with the unweighted UniFrac distance metric. Significant differences between groups were found for the microbial composition of feces (Clostridiaceae, Turicibacteriaceae for family level and Clostridium , Dorea , SMB53 and Turicibacter for genus level), blood (Tenericutes for phylum level and Mycoplasma for genus level) and milk (OD1 and Proteobacteria for phylum level, Enterobacteriaceae and Moraxallaceae for family level and Olsenella and Rhodococcus for genus level). The beta biodiversity of feces and blood did not change between groups. Significant differences ( p < 0.05) were observed between the beta diversity in milk of G group and Y group and between Y group and R group. The number of taxa in common between feces, blood and milk were 8 at a phylum, 19 at a family and 15 at a genus level. From these results, the bacterial crossing from gut to milk in cows was not confirmed but remained hypothetical and deserves further investigation.

Genomic selection (GS) reports on milk fatty acid (FA) profiles have been published quite recently and are still few despite this trait represents the most important aspect of milk nutritional and sensory quality. Reasons for this can be found in the high costs of phenotype recording but also in issues related to its nature of complex trait constituted by multiple genetically correlated variables with low heritabilities. One possible strategy to deal with such constraint is represented by the use of dimension reduction methods. We analysed 40 individual FAs from Italian Brown Swiss, Holstein and Simmental milk through multivariate factor analysis (MFA) to study the genetics of milk FA-related latent variables (factors) and assess their potential use in breeding. A total of nine factors were obtained, and their genetic parameters were inferred under a Bayesian framework using two statistical approaches: the classical pedigree best linear unbiased prediction (ABLUP) and the single-step genomic BLUP (ssGBLUP). The resulting factorial solutions were able to represent groups of FAs with common origin and function and can be considered concise pathway-level phenotypes. The heritability (h 2 ) values showed relevant variations across different factors in each breed (0.03 ≤ h 2  ≤ 0.38). The accuracies of breeding values predicted were low to high, ranging from 0.13 to 0.72 and from 0.18 to 0.74 considering the pedigree and the genomic model, respectively. The gain in accuracy in genetic prediction due to the addition of genomic information was ~30% and ~5% in validation and training groups respectively, confirming the contribution of genomic information in yielding more accurate predictions compared to the traditional ABLUP methodology. Our results suggest that MFA in combination with GS can be a valuable tool in dairy cattle breeding and deserves to be further investigated for use in future breeding programs to improve cow's milk FA-related traits., (© 2020 John Wiley & Sons Ltd.)

Stress in livestock reduces productivity and is a welfare concern. At a physiological level, stress is associated with the activation of inflammatory responses and increased levels of harmful reactive oxygen species. Biomarkers that are indicative of stress could facilitate the identification of more stress-resilient animals. We examined twenty-one metabolic, immune response, and liver function biomarkers that have been associated with stress in 416 Italian Simmental and 436 Italian Holstein cows which were genotyped for 150K SNPs. Single-SNP and haplotype-based genome-wide association studies were carried out to assess whether the variation in the levels in these biomarkers is under genetic control and to identify the genomic loci involved. Significant associations were found for the plasma levels of ceruloplasmin ( Bos taurus chromosome 1-BTA1), paraoxonase (BTA4) and γ-glutamyl transferase (BTA17) in the individual breed analysis that coincided with the position of the genes coding for these proteins, suggesting that their expression is under cis-regulation. A meta-analysis of both breeds identified additional significant associations with paraoxonase on BTA 16 and 26. Finding genetic associations with variations in the levels of these biomarkers suggests that the selection for high or low levels of expression could be achieved rapidly. Whether the level of expression of the biomarkers correlates with the response to stressful situations has yet to be determined.

Protected Designation of Origin (PDO) dry-cured hams production is greatly dependent on raw meat quality. This study was performed to identify genetic markers associated with the quality of dry-cured ham. Carcass traits of 229 heavy pigs belonging to three commercial genetic lines were registered (weight, EUROP classification). Phenotypic traits ( Semimembranosus muscle ultimate pH, ham weight and lean meat content, adsorbed salt) of the corresponding thighs, undergone PDO ham process in three different plants, were measured, using a fast and non-invasive technology. Green ham weight and lean meat percentage influenced the estimated salt content and the weight loss during salting, even if the processing plant greatly affected the variability of the measured ham traits. The genomic data were obtained with the GeneSeek Genomic Profiler (GGP) 70k HD Porcine Array, using the slaughter day and the sex of the animals in the statistical analyses. The phenotypic traits were associated with the genotypes through GenAbel software. The results showed that 18 SNPs located on nine porcine chromosomes were found to be associated with nine phenotypic traits, mainly related to ham weight loss during salting. New associations were found between markers in the genes Neural Precursor Cell Expressed Developmentally Down-Regulated 9 ( NEDD9 , SSC7), T-Cell Lymphoma Invasion and Metastasis 2 ( TIAM2 , SSC1), and the ham quality traits. After validation, these SNPs may be useful to improve the quality of thighs for the production of PDO dry-cured hams.

The characterization of the microbial population in different compartments of the organism, such as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field have already investigated the fecal microbiome in healthy or sick subjects; however, the methodologies used in the different laboratories and the limited number of animals recruited in each experiment do not allow a straight comparison among published results. Previously, our research focused on the characterization of the microbial taxa variability in 340 fecal samples from 132 healthy dogs, collected serially from several in-house experiments. The results supported the responsiveness of microbiota to dietary and sex factors and allowed us to cluster dogs with high accuracy. For the present study, intestinal and blood microbiota of healthy dogs from different breeds, genders, ages and food habits were collected, with three principal aims: firstly, to confirm the results of our previous study regarding the fecal microbiome affected by the different type of diet; secondly, to investigate the existence of a blood microbial population, even in heathy subjects; and thirdly, to seek for a possible connection between the fecal and the blood microbiota. Limited researches have been published on blood microbiota in humans, and this is the first evidence of the presence of a bacterial population in the blood of dogs. Moreover, gut and blood microbiota can discriminate the animals by factors such as diet, suggesting some relationship between them. These preliminary results make us believe in the use of the blood microbiome for diagnostic purposes, such as researching and preventing gut inflammatory diseases.

A dietary intervention study was assessed to determine if different sources of starch in homemade diets could significantly modify fecal microbiome of dogs. Twenty-seven adult dogs were enrolled and fed a diet based on a mixture of rice and pasta with fresh raw meat (CD). After 90 d, 8 dogs continued to receive CD diet, 10 dogs received a diet made of a raw meat and a complementary food with rice as the main source of starch (B1), and 9 dogs were fed a diet with the same raw meat and a complementary food with potato as the main source of starch (B2). Samples of feces were collected from each dog in the mornings at the beginning of the study and after 15 d and analyzed for pH, ammonia N (N-NH 3 ) and total N, short chain fatty acids (SCFA) and lactic acid. Relative abundance of fecal microbiota was assessed by sequencing and annotating the V3-V4 regions of the 16S rRNA. Total starch intake was similar between diets but differed in the in vitro rate digestion and in the resistant starch, which was higher in B2 than in B1 and CD diets. Dogs fed B2 diet showed lower ( P  < 0.05) N-NH 3 and pH but higher ( P  < 0.05) molar proportion of lactic acid. Linear discriminant analysis of the genera relative abundances indicated a significant ( P  < 0.01) increase of SMB53 genus at the end of the study in B1 diet and of Megamonas genus in B1 and B2 diets in comparison to CD diet. These results suggest that changes of starch source in a raw meat-based diet have limited effects on fecal microbiome in healthy dogs, but underline a high variability of microbiota among dogs., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper., (© 2020 Chinese Association of Animal Science and Veterinary Medicine. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.)

Milk fatty acid (FA) profile is a clear example of complex and multiple correlated traits whose genetic basis is difficult to assess. Although genome-wide association (GWA) studies have been successful in the identification of significant genetic variants for complex traits, when correlated phenotypes are analysed separately, the outcomes are difficult to compare and interpret in a metabolic context. Here, we performed a multivariate factor analysis (MFA) on Italian Simmental and Italian Holstein milk fat profiles to extract latent unobserved factors able to explain correlation structure and common metabolic function among different FAs. Individual factor scores obtained by MFA were used to perform a single-SNP based GWA. In both breeds, MFA was able to extract ten latent factors with specific biological meaning, notably: de novo synthesis, desaturation activity and biohydrogenation. The GWA result confirmed the increased power of joint association analysis on multiple correlated traits and allowed us to identify major candidate genes with well-documented function consistent with the metabolic classification of factors obtained, such as DGAT1, FASN and SCD.

The characterization of the microbial population of many niches of the organism, as the gastrointestinal tract, is now possible thanks to the use of high-throughput DNA sequencing technique. Several studies in the companion animals field already investigated faecal microbiome in healthy or affected subjects, although the methodologies used in the different laboratories and the limited number of animals recruited in each experiment does not allow a straight comparison among published results. In the present study, we report data collected from several in house researches carried out in healthy dogs, with the aim to seek for a variability of microbial taxa in the faeces, caused by factors such as diet and sex. The database contains 340 samples from 132 dogs, collected serially during dietary intervention studies. The procedure of samples collection, storage, DNA extraction and sequencing, bioinformatic and statistical analysis followed a standardized pipeline. Microbial profiles of faecal samples have been analyzed applying dimensional reduction discriminant analysis followed by random forest analysis to the relative abundances of genera in the feces as variables. The results supported the responsiveness of microbiota at a genera taxonomic level to dietary factor and allowed to cluster dogs according this factor with high accuracy. Also sex factor clustered dogs, with castrated males and spayed females forming a separated group in comparison to intact dogs, strengthening the hypothesis of a bidirectional interaction between microbiota and endocrine status of the host. The findings of the present analysis are promising for a better comprehension of the mechanisms that regulate the connection of the microorganisms living the gastrointestinal tract with the diet and the host. This preliminary study deserves further investigation for the identification of the factors affecting faecal microbiome in dogs., Competing Interests: DL and SDM are employees of ARGO Open Lab Platform for Genome sequencing, AREA Science Park, Padriciano, Trieste, Italy. Remuneration was not received for drafting, reviewing, or approving the manuscript for publication. None of these declarations alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Recent advances on milk exosomes (EXO), cargoes in cell-cell communication, explored their role within and between individuals, including in dairy species. The potential use of EXO as biomarkers of disease and metabolic conditions adds significant interest to the study of EXO in milk. Although several researches have been carried out on circulating miRNA in the milk, less information is available about milk-derived exosomal miRNAs, which are stable over time and resistant to digestion and milk processing. EXO are taken up by recipient cells through specific mechanisms, which enable the selective delivery of cargoes. This suggests that EXO cargoes can be used as biomarkers of health. Nevertheless, methodological limitations and potential applications of milk EXO in dairy ruminants must be considered. The paucity of studies that associate the EXO cargo to specific challenges deserves further investigations to unravel the variation of miRNA and proteins cargo in relation to metabolic imbalance and infectious disease of the mammary gland.

Dairy cattle health, wellbeing and productivity are deeply affected by stress. Its influence on metabolism and immune response is well known, but the underlying epigenetic mechanisms require further investigation. In this study, we compared DNA methylation and gene expression signatures between two dairy cattle populations falling in the high- and low-variant tails of the distribution of milk cortisol concentration (MC), a neuroendocrine marker of stress in dairy cows. Reduced Representation Bisulfite Sequencing was used to obtain a methylation map from blood samples of these animals. The high and low groups exhibited similar amounts of methylated CpGs, while we found differences among non-CpG sites. Significant methylation changes were detected in 248 genes. We also identified significant fold differences in the expression of 324 genes. KEGG and Gene Ontology (GO) analysis showed that genes of both groups act together in several pathways, such as nervous system activity, immune regulatory functions and glucocorticoid metabolism. These preliminary results suggest that, in livestock, cortisol secretion could act as a trigger for epigenetic regulation and that peripheral changes in methylation can provide an insight into central nervous system functions.

Several studies have underlined the interplay among host-microbiome and pathophysiological conditions of animals. Research has also focused specifically on whether and how changes in the gut microbiome have provoked the occurrence of pathological phenomena affecting cartilage and joints in humans and in laboratory animals. Here, we tried to evaluate the relationship between the gut microbiome and the hip and elbow arthritis in owned dogs. The study included 14 dogs suffering from chronic arthritis (AD) and 13 healthy dogs (HD). After the first visit and during the period of the study, the dogs, under the supervision of the owner, were fed a semi-moist complete diet supplemented with omega 3 fatty acids. Feces and blood samples were collected in the clinic at the first visit (T0) and after days (T45). The plasma C-reactive protein (CRP) was higher, and the serum vitamin B12 and folate concentrations were lower ( p < 0.05) in the AD group in comparison to the HD group. Data of the fecal microbiome showed that the relative abundances of the genus Megamonas were higher in AD ( p < 0.001), while the relative abundance of the families Paraprevotellaceae, Porphyromonadaceae, and Mogibacteriaceae was significantly lower in comparison to HD. The results of the study identified several bacterial groups that differed significantly in the fecal microbiome between healthy and diseased dogs. If the observed differences in fecal bacterial composition predispose dogs to hip and elbow arthritis or if these differences reflect a correlation with these conditions deserves further investigation.

Recent advances in ruminants' milk-derived exosomes (EXO) have indicated a role of microRNAs (miRNAs) in cell-to-cell communication in dairy ruminants. The miRNAs EXO retain peculiar mechanisms of uptake from recipient cells, which enables the selective delivery of cargos, with a specific regulation of target genes. Although many studies have been published on the miRNAs contained in milk, less information is available on the role of miRNAs EXO, which are considered stable over time and resistant to digestion and milk processing. Several miRNAs EXO have been implicated in the cellular signaling pathway, as in the regulation of immune response. Moreover, they exert epigenetic control, as extenuating the expression of DNA methyltransferase 1. However, the study of miRNAs EXO is still challenging due to the difficulty of isolating EXO. In fact, there are not agreed protocols, and different methods, often time-consuming, are used, making it difficult to routinely process a large number of samples. The regulation of cell functions in mammary glands by miRNAs EXO, and their applications as genomic markers in livestock, is presented., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Differential leukocyte count (DSCC) in milk is considered important to improve knowledge of udder immune response. The investigations on milk DSCC were limited by the techniques available until recently, when a high-throughput tool to perform DSCC opened the way to explore these factors in rapid and economically sustainable ways. We hypothesized that DSCC alone does not fully describe the pattern of these cells, since the total amount is also influenced by milk yield and SCC. Therefore, this study was designed to describe DSCC and total amount of different leukocytes in milk during the course of lactation in cows differing in parity and in levels of SCC. This study considered 17,939 individual milk tests from 12 dairy herds in Lombardy Region, where DCC testing was applied in the period of February 2018-December 2019 (23 months). The samples were divided into two subsets-"healthy" (HS) with SCC ≤200,000 cells/mL and "inflamed" (IS) with SCC >200,000 cells/mL. Cow in HS have a P + LT average between 5.0 × 10 8 and 3.0 × 10 9 cells. In IS cows, the values were 1.6 × 10 10 and 2.5 × 10 10 . Therefore, the presence of a well-defined inflammatory process increased the overall amount of polymorphonuclear neutrophils (PMN) and lymphocytes (LYM) of 1 log, from 1 × 10 9 to 1 × 10 10 . The assessment of the total amount of PMN and LYM, to our knowledge, have never been reported in scientific literature; the values observed may be proposed as benchmarks for studies on udder immune response. When data were analyzed by days in milk (DIM), they showed that cows in first and second lactation have a significantly lower amount of PMN + LYM, when compared to cows in third and higher lactation. However, these differences are numerically not very large (7%), and suggest that, in healthy animals, the number of immune cells is kept as constant as possible. In IS, the analysis of trends based on DIM showed that both DSCC and P + LT have a significant negative trend. These data suggest that only in this group, the presence of high SCC as lactation proceeds is associated with a progressive increase in the number of macrophages. To the best of our knowledge, this is the first study describing the pattern of DSCC and the total amount of PMN + LYM in relation to parity, days in milk, and SCC, and it may be considered as the first contribution in the investigation on mammary gland immune response by the means of differential cell counts in milk.

Several studies on the interaction between gut microbiota and diets, including prebiotics, have been reported in dogs, but no data are available about the effects of dietary administration of grape proanthocyanidins. In the study, 24 healthy adult dogs of different breeds were recruited and divided in 3 groups of 8 subjects each. A group was fed with a control diet (D0), whilst the others were supplemented with 1 (D1) or 3 (D3) mg/kg live weight of grape proanthocyanidins. Samples of feces were collected at the beginning and after 14 and 28 days for microbiota, short chain fatty acid, and lactic acid analysis. Serotonin and cortisol were measured in saliva, collected at the beginning of the study and after 28 days. A significantly higher abundance ( p < 0.01) of Enterococcus and Adlercreutzia were observed in D0, whilst Escherichia and Eubacterium were higher in D1. Fusobacterium and Phascolarctobacterium were higher ( p < 0.01) in D3. Salivary serotonin increased ( p < 0.01) at T28 for D1 and D3 groups but cortisol did not vary. Proanthocyanidins administration influenced the fecal microbiota and neuroendocrine response of dogs, but a high variability of taxa was observed, suggesting a uniqueness and stability of fecal microbiota related to the individual.

This study examined microRNA (miRNAs) expression and regulatory patterns from milk exosomes of cows experienced group relocation, a common husbandry practice during the lactation period and used as a spontaneous model of stress. Total RNA from milk exosome samples was collected from 3 cows that showed an increased milk cortisol (HRC) after relocation (T2 vs T1) and 3 cows that did not show cortisol increase (LRC). A total of 69 known miRNAs were identified. Thirteen miRNAs were consistently down-regulated at T2 in comparison to T1, such as miR-2904-1, miR-142, miR-2284x and miR-30b-3p. Only two miRNAs, miR-2284z and miR-146a were significantly different between LRC and HRC group. Functional enrichment analyses highlighted that glucocorticoid receptor signaling and neurotrophic factor mediated TRK receptor signaling were among the biological pathways affected by differentially expressed miRNA target genes. Mir-135a-5p and miR-320a were involved in both biological pathways. miR-142-5p, miR-142-3p, miR-30b-5p and miR-320a shared the same target genes belonging to the RAS superfamily of small GTP-binding proteins (RAC1, RAP1A and RASA1), involved in neurotrophin-mediated cell survival. MiR-142, miR-135 and miR-320a in milk exosomes were the most responsive to group relocation of cows., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In the veterinary sector, many papers deal with the relationships between inflammation and oxidative stress. However, few studies investigate the mechanisms of action of oxidised molecules in the regulation of immune cells. Thus, authors often assume that these events, sometime leading to oxidative stress, are conserved among species. The aim of this review is to draw the state-of-the-art of the current knowledge about the role of oxidised molecules and dietary antioxidant compounds in the regulation of the immune cell functions and suggest some perspectives for future investigations in animals of veterinary interest.

Background: Dietary intervention studies are required to deeper understand the variability of gut microbial ecosystem in healthy dogs under different feeding conditions and to improve diet formulations. The aim of the study was to investigate in dogs the influence of a raw based diet supplemented with vegetable foods on faecal microbiome in comparison with extruded food., Methods: Eight healthy adult Boxer dogs were recruited and randomly divided in two experimental blocks of 4 individuals. Dogs were regularly fed a commercial extruded diet (RD) and starting from the beginning of the trial, one group received the raw based diet (MD) and the other group continued to be fed with the RD diet (CD) for a fortnight. After 14 days, the two groups were inverted, the CD group shifted to the MD and the MD shifted to the CD, for the next 14 days. Faeces were collected at the beginning of the study (T0), after 14 days (T14) before the change of diet and at the end of experimental period (T28) for DNA extraction and analysis of metagenome by sequencing 16SrRNA V3 and V4 regions, short chain fatty acids (SCFA), lactate and faecal score., Results: A decreased proportion of Lactobacillus, Paralactobacillus (P < 0.01) and Prevotella (P < 0.05) genera was observed in the MD group while Shannon biodiversity Index significantly increased (3.31 ± 0.15) in comparison to the RD group (2.92 ± 0.31; P < 0.05). The MD diet significantly (P < 0.05) decreased the Faecal Score and increased the lactic acid concentration in the feces in comparison to the RD treatment (P < 0.01). Faecal acetate was negatively correlated with Escherichia/Shigella and Megamonas (P < 0.01), whilst butyrate was positively correlated with Blautia and Peptococcus (P < 0.05). Positive correlations were found between lactate and Megamonas (P < 0.05), Escherichia/Shigella (P < 0.01) and Lactococcus (P < 0.01)., Conclusion: These results suggest that the diet composition modifies faecal microbial composition and end products of fermentation. The administration of MD diet promoted a more balanced growth of bacterial communities and a positive change in the readouts of healthy gut functions in comparison to RD diet.

The aim of this study was to investigate the adipocyte size and fate in subcutaneous fat (scAT) of cows diverging for genetic merit at mid lactation stage, when anabolic activity increases and animals are in a state of positive energy balance. Twenty mid lactation cows (180±20days in milk) grouped according to the Estimated Breeding Values (EBV) for milk yield in plus (EBVp) and minus (EBVm) variants were selected. Average of adipocytes area, proliferation and apoptotic labelling index as well as DLK-1 expression, a marker of pre-adipocytes, were immunohistochemically evaluated in scAT biopsies. In EBVp cows, the BCS was lower (P<0.01) whereas milk yield, protein, fat yield (P<0.001) and plasma free fatty acid concentration (P<0.05) were higher. The scAT of EBVp cows showed a significantly (P<0.001) higher frequency between 500 and 3000μm 2 classes in comparison to EBVm cows, that showed a significantly (P<0.01) higher apoptotic labeling index. The immunohistochemical reaction showed DLK-1 positivity in scAT of EBVp cows. Taking together, the data indicate a link between milk yield genetic merit of cows, scAT morphology and function, suggesting greater dynamics and metabolic flexibility in EBVp cows., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aim of the study reported in this Research Communication was to analyse the variations of milk cortisol concentrations in response to the relocation of dairy cows between production groups. Milk cortisol measured during 3 consecutive days did not vary significantly in cows without environmental perturbation. However, relocation of cows caused a significant increase of cortisol in milk starting from the first milking after the group change. This suggests that cortisol in milk can be a suitable biomarker to assess the HPA response of dairy cows to a short/medium-term environmental challenge.

The study was conducted to investigate the effects of dietary administrations of four nutraceuticals in dogs. Seventy four dogs were enrolled in the trials, 24 healthy dogs were fed with a control diet (CT) and the experimental groups received for 60days the same diet supplemented with nutraceuticals, namely Echinacea angustifolia (EA, 0.10mg/kg live weight as echinacoside; 14 dogs), Vaccinium myrtillus (VM, 0.20mg/kg live weight as anthocyanidin, 13 dogs), Curcuma longa (CL, 6.60mg/kg live weight as curcumin, 18 dogs with arthrosis), and Sylibum marianum (SM, 1.5mg/kg live weight as sylibin, 8 dogs with hepatopathy). Dogs were weighted at the beginning of study and blood samples were collected at the beginning (T0) and at the end (T60) of the study. VM significantly down regulated TNF, CXCL8, NFKB1 and PTGS2 and decreased plasma ceruloplasmin (CuCp). The activity of EA was evidenced by the significant decrease of TNF and NFKB1 expression and CuCp levels and by the increase of plasma Zn. Administration of CL caused a significant decrease of CuCp and increase of Zn and a down regulation of TNF, CXCL8, NFKB1 and PTGS2, corroborating the anti-inflammatory action of curcuminoids. After 60days of treatment with SM, plasma ALT/GPT activity was reduced and paraoxonase was increased, supporting the antioxidant activity of silymarin, also confirmed by the significant up regulation of SOD2. Results indicated that nutraceutical administrations in dogs can be an interesting approach to modulate immune response in order to improve health condition of animals., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Hydroxytyrosol has various pharmacological properties, including anti-oxidative stress and anti-inflammatory activities, preventing hyperglycemia, insulin resistance, and the metabolic syndrome. The present study is focused on the anti-adipogenic and lipolytic activity of hydroxytyrosol on primary human visceral adipocytes. Pre-adipocytes were analyzed after 10 (P10) and 20 (P20) days of treatment during differentiation and after 7 (A7) days of treatment when they reached mature shape. The treatment with hydroxytyrosol extract significantly (P < 0.001) increased apoptosis in P10 and P20 cells in comparison to control and A7 cells; significantly (P < 0.001) reduced triglyceride accumulation in P20 cells compared to P10 and control cells; and significantly (P < 0.001) increased lipolysis in P20 cells in comparison to control cells and A7 mature adipocytes. Hydroxytyrosol-treated P20 cells significantly (P < 0.05) increased expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT3A, SFRP5, HES1, and SIRT1. In contrast, genes involved in promoting adipogenesis such as LEP, FGF1, CCND1, and SREBF1 were significantly down-regulated by hydroxytyrosol treatment. These data suggest that hydroxytyrosol promotes lipolysis and apoptotic activity in primary human visceral pre-adipocytes during differentiation and does not affect already mature adipocytes., (© 2016 by the Society for Experimental Biology and Medicine.)

Several natural compounds exhibit strong capacity for decreasing triglyceride accumulation, enhancing lipolysis and inducing apoptosis. The present study reports the anti-adipogenic effects of Silybum marianum (SL), Citrus aurantium (CA), Taraxacum officinale (TO), resveratrol (RE), Curcuma longa (CU), caffeine (CF), oleuropein (OL) and docosahexaenoic acid (DHA) in reducing differentiation and increasing lipolysis and apoptosis. Analyses were performed on human primary visceral pre-adipocytes after 10 (P10) and 20 (P20) days of treatment during differentiation and on mature adipocytes after 7 days of treatment (A7). The percentage of apoptosis induced by TO extract in P10 and P20 cells was significantly higher than that induced by all other compounds and in CTRL cells. Triglyceride accumulation was significantly lower in cells treated with DHA, CF, RE in comparison to cells treated with OL and in CTRL cells. Treatments with CF, DHA and OL significantly incremented lipolysis in P20 cells in comparison to other compounds and in CTRL cells. On the contrary, the treatment of A7 cells with OL, CA and TO compounds significantly increased cell lipolysis. The addition of CF in differentiating P20 pre-adipocytes significantly increased the expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT1, WNT3A, SFRP5, and DLK1. Genes involved in promoting adipogenesis such as CCND1, CEBPB and SREBF1 were significantly down-regulated by the treatment. The screening of bioactive compounds for anti-adipogenic effects showed that in differentiating cells TO extract was the most effective in inducing apoptosis and CF and DHA extracts were more efficient in inhibition of differentiation and in induction of cell lipolysis.

Background: Whether the measurement of cortisol in dairy cows can be used as a biomarker of adverse environmental or pathophysiological conditions is still under of scientific debate. In these situations, several systems mainly the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and the immune system are recruited to reestablish homeostasis. A first aim of the present study was to compare milk and blood cortisol concentrations and to consider its variability in milk in relation to farm, milk yield and days in milk. A second study investigates the effects of breed, class of somatic cell count (SCC) and farm on milk cortisol levels in a larger number of cows and farms, with the aim to validate the results obtained in the pilot study., Methods: For study 1, 135 cows were sampled from 2 Italian Simmental and 2 Italian Holstein commercial farms, whilst in the second study, 542 cows were sampled from 6 commercial farms of Italian Simmental and 499 cows from 4 commercial farms of Italian Holstein., Results: In study 1, the values of cortisol content in milk were significantly higher in Holstein than Simmental cows. Significant differences between farms were observed for milk and plasma cortisol concentrations. Cortisol content in milk was not correlated to plasma content in study 1 and the mean milk to plasma cortisol ratio was about 1:30. In study 2, for Holstein cows, significantly higher values of milk cortisol in comparison to Simmental cows was reported. A significant effect of class of SCC was observed, cows belonging to class 3 (SCC higher than 400.000/ml) showed the highest mean values of milk cortisol. The farm effect was significant also in the study 2, confirming the results obtained in the first study., Conclusions: Milk can be considered a preferential site of sampling in dairy cows to point out short term stimulation of the hypothalamic-pituitary-adrenal axis. Further studies are needed to investigate the physiological basis of the relationship between milk cortisol content and breed, milk yield and SCC to ascertain the relevance of milk cortisol to monitor the healthy status of mammary gland.

The discovery of new protein-coding DNA variants related to carcass traits is very important for the Italian pig industry, which requires heavy pigs with higher thickness of subcutaneous fat for Protected Designation of Origin (PDO) productions. Exome capture techniques offer the opportunity to focus on the regions of DNA potentially related to the gene and protein expression. In this research a human commercial target enrichment kit was used to evaluate its performances for pig exome capture and for the identification of DNA variants suitable for comparative analysis. Two pools of 30 pigs each, crosses of Italian Duroc X Large White (DU) and Commercial hybrid X Large White (HY), were used and NGS libraries were prepared with the SureSelectXT Target Enrichment System for Illumina Paired-End Sequencing Library (Agilent). A total of 140.2 M and 162.5 M of raw reads were generated for DU and HY, respectively. Average coverage of all the exonic regions for Sus scrofa (ENSEMBL Sus&#95;scrofa.Sscrofa10.2.73.gtf) was 89.33X for DU and 97.56X for HY; and 35% of aligned bases uniquely mapped to off-target regions. Comparison of sequencing data with the Sscrofa10.2 reference genome, after applying hard filtering criteria, revealed a total of 232,530 single nucleotide variants (SNVs) of which 20.6% mapped in exonic regions and 49.5% within intronic regions. The comparison of allele frequencies of 213 randomly selected SNVs from exome sequencing and the same SNVs analyzed with a Sequenom MassARRAY® system confirms that this "human-on-pig" approach offers new potentiality for the identification of DNA variants in protein-coding genes.

Currently, there are no reports of neurotrophins in adipose tissue of cows. The distribution of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and their high-affinity tyrosine kinase receptors TrkA and TrkB, was investigated by immunohistochemical method in the subcutaneous adipose tissue of cow at mid-lactation. Results revealed the localization of NGF and BDNF along the plasma membrane and cytoplasm of adipocytes. Neurotrophin receptors TrkA and TrkB showed moderate and strong positive staining in adipocytes, respectively. The expression of NGF, BDNF, TRKB--but not of TRKA--was also confirmed at transcriptional level by RT-PCR analyses. Considering the involvement of BDNF on fat metabolism and of NGF on activation of the sympathetic response in human and rodents, these neurotrophins could be related to lipogenesis and lipolysis occurring during lactation in cows. The local production of these neurotrophins supports their potential paracrine function for the regulation of adipocyte activity and deserve further investigations., (Copyright © 2015. Published by Elsevier Ltd.)

Genome-wide association studies (GWAS) have been widely applied to disentangle the genetic basis of complex traits. In cattle breeds, classical GWAS approaches with medium-density marker panels are far from conclusive, especially for complex traits. This is due to the intrinsic limitations of GWAS and the assumptions that are made to step from the association signals to the functional variations. Here, we applied a gene-based strategy to prioritize genotype-phenotype associations found for milk production and quality traits with classical approaches in three Italian dairy cattle breeds with different sample sizes (Italian Brown n = 745; Italian Holstein n = 2058; Italian Simmental n = 477). Although classical regression on single markers revealed only a single genome-wide significant genotype-phenotype association, for Italian Holstein, the gene-based approach identified specific genes in each breed that are associated with milk physiology and mammary gland development. As no standard method has yet been established to step from variation to functional units (i.e., genes), the strategy proposed here may contribute to revealing new genes that play significant roles in complex traits, such as those investigated here, amplifying low association signals using a gene-centric approach., (© 2015 Stichting International Foundation for Animal Genetics.)

The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement., (© 2015 by the Society for Experimental Biology and Medicine.)

Rhodiola rosea (Rro) has been reported to have various pharmacological properties, including anti-fatigue, anti-stress and anti-inflammatory activity. It is also known to improve glucose and lipid metabolism, but the effects of Rhodiola rosea on adipocyte differentiation and metabolism are not still elucidated. In this study the anti-adipogenic and lipolytic activity of two extracts of Rhodiola rosea, containing 3% salidroside (RS) or 1% salidroside and 3% rosavines (RR) on primary human visceral adipocytes was investigated. Pre-adipocytes were analyzed after 10 and 20 days of treatment during differentiation and after 7 days of treatment when they reached mature shape. The RS extract significantly induced higher apoptosis and lipolysis in comparison to control cells and to RR extract. In contrast, RR extract significantly reduced triglyceride incorporation during maturation. Differentiation of pre-adipocytes in the presence of RS and RR extracts showed a significant decrease in expression of genes involved in adipocyte function such as SLC2A4 and the adipogenic factor FGF2 and significant increase in expression of genes involved in inhibition of adipogenesis, such as GATA3, WNT3A, WNT10B. Furthermore RR extract, in contrast to RS, significantly down-regulates PPARG, the master regulator of adipogenesis and FABP4. These data support the lipolytic and anti-adipogenetic activity of two different commercial extracts of Rhodiola rosea in primary human visceral pre-adipocytes during differentiation.

Background: Arctium lappa (AL), Camellia sinensis (CS), Echinacea angustifolia, Eleutherococcus senticosus, Panax ginseng (PG), and Vaccinium myrtillus (VM) are plants traditionally used in many herbal formulations for the treatment of various conditions. Although they are well known and already studied for their anti-inflammatory properties, their effects on H2O2-stimulated macrophages are a novel area of study., Materials and Methods: Cell viability was tested after treatment with increasing doses of H2O2 and/or plant extracts at different times of incubation to identify the optimal experimental conditions. The messenger (m)RNA expression of TNFα, COX2, IL1β, NFκB1, NFκB2, NOS2, NFE2L2, and PPARγ was analyzed in macrophages under H2O2 stimulation. The same genes were also quantified after plant extract treatment on cells pre-stimulated with H2O2., Results: A noncytotoxic dose (200 μM) of H2O2 induced active mRNA expression of COX2, IL1β, NFE2L2, NFκB1, NFκB2, NOS2, and TNFα, while PPARγ was depressed. The expression of all genes tested was significantly (P<0.001) regulated by plant extracts after pre-stimulation with H2O2. COX2 was downregulated by AL, PG, and VM. All extracts depressed IL1β expression, but upregulated NFE2L2. NFκB1, NFκB2, and TNFα were downregulated by AL, CS, PG, and VM. NOS2 was inhibited by CS, PG, and VM. PPARγ was decreased only after treatment with E. angustifolia and E. senticosus., Conclusion: The results of the present study indicate that the stimulation of H2O2 on RAW267.4 cells induced the transcription of proinflammatory mediators, showing that this could be an applicable system by which to activate macrophages. Plant extracts from AL, CS, PG, and VM possess in vitro anti-inflammatory activity on H2O2-stimulated macrophages by modulating key inflammation mediators. Further in vitro and in vivo investigation into molecular mechanisms modulated by herbal extracts should be undertaken to shed light on the development of novel modulating therapeutic strategies.

Although the biological activities of Arctium lappa (burdock) have been already investigated in human and other species, data evaluating the molecular mechanisms have not been reported in the dog. In this study we analyzed for the first time the effect of a root extract of burdock on molecular responses in canine dermal fibroblasts with H2O2 stimulation (H group), with burdock treatment (B group) and with H2O2 stimulation and burdock treatment (BH group), using RNAseq technology. Differentially expressed genes (P<0.05) of H, B and BH groups in comparison to the untreated sample (negative control, C group) were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). The expression profile of canine dermal fibroblasts treated with burdock extract with or without H2O2 stimulation, showed an up-regulation of mitochondrial superoxide dismutase (SOD2), disheveled 3 (DVL3) and chondroitin sulfate N-acetylgalactosaminyltransferase 2 (CSGALNACT2). The data suggested that burdock has implications in cell adhesion and gene expression with the modulation of Wnt/β catenin signaling and Chondroitin Sulphate Biosynthesis that are particularly important for the wound healing process., (© 2013 Elsevier B.V. All rights reserved.)

The dietary effect of non-steroidal anti-inflammatory drug (NSAID) or curcumin on the gene expression of peripheral white blood cells in osteoarthritis (OA) affected dogs was investigated using a 44K oligo microarray. Two groups of OA dogs and one group of healthy dogs (6 dogs each) were clinically evaluated and blood was sampled before (T0) and after 20days (T20) of dietary administration of NSAID (NSAID group) or curcumin (CURCUMIN group). Differentially expressed genes (P<0.05) in comparison to the control group were identified with MeV software and were functional annotated and monitored for signaling pathways and candidate biomarkers using the Ingenuity Pathways Analysis (IPA). After 20days of treatment, the differentially expressed transcripts significantly (P<0.05) decreased from 475 to 173 in NSAID group and from 498 to 141 in CURCUMIN group. Genes involved in "inflammatory response" and in "connective tissue development and function" dramatically decreased at T20. Other genes, included in "cellular movement", "cellular compromise" and "immune cell trafficking", were differentially expressed at T0 but not at T20 in both groups. Specific molecular targets of CURCUMIN, not observed for NSAID, were the IkB up regulation in the "TNRF1 signaling pathway" and IL18 down regulation in the "role of cytokines in mediating communication between immune cells". The activity of CURCUMIN was also evidenced from the inhibition of macrophages proliferation (HBEGF), related to a strong down regulation of TNFα and to activation of fibrinolysis (SERPINE1). The results would suggest that curcumin offers a complementary antinflammatory support for OA treatment in dogs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Impaired neutrophil function has been associated with increased infectious diseases in ruminants. Attachment of neutrophils to endothelium and superoxide production is critical features of their immune activity. Once the infection is cleared, programmed cell death ensures the rapid resolution of inflammation. To develop new natural therapeutics for ruminants, standard extracts of Echinacea angustifolia (Polinacea), Butea frondosa and Curcuma longa (Curcuvet) were first evaluated on ovine neutrophil functions. Curcuvet strongly reduced PMA-stimulated adhesion and superoxide production. Polinacea and B. frondosa extract also reduced these functions, but with less efficacy than Curcuvet. We analyzed the effect of extracts on spontaneous apoptosis and gene expression in neutrophils aged in vitro for up to 22h. IL8 is critical for neutrophil recruitment and the immune response; Bcl2-related proteins, Bcl2A1 and Bax, are key regulators of neutrophil fate. Spontaneous apoptosis strongly increased in ovine neutrophils cultured for 22h (T22), accompanied by an upregulation of IL8 and a decreased Bcl2A1:Bax ratio. Curcuvet stimulated spontaneous apoptosis and inhibited IL8 and Bcl2A1 gene expression at T22, whereas Polinacea and B. frondosa extract inhibited spontaneous apoptosis and stimulated IL8 expression at T22. These results suggest that Curcuvet has antiinflammatory activity, whereas Polinacea and B. frondosa have an immunomodulatory action on sheep neutrophils.

Neutrophils play an essential role in host defense and inflammation. Plants have long been used to improve the immune function, but for most of them specific investigations on animal health are lacking. In the present study, water and hydroethanolic extracts from 11 plant wastes have been screened on immune responses of ovine neutrophils. Eight sheep clinically healthy, not lactating, non-pregnant were selected and used for the experiment. Freshly isolated neutrophils were incubated with the extracts of the residues at increasing doses, and then they were tested for adhesion and superoxide production induced with PMA. The residues of Larix decidua, Thymus vulgaris, Salix alba, Sinupret, Helianthus annuus, Mangifera indica modulated the neutrophil immune functions, moreover, Larix decidua, Thymus vulgaris and Salix alba presented the highest anti-inflammatory activity.

The volatile fraction of San Daniele hams was evaluated in barrows and gilts belonging to two commercial breeds. A group of 120 pigs, reared under the same conditions, were slaughtered, and the right ham seasoned/cured for 12 months, in accordance with the San Daniele Consortium protocol. At the end of ripening, 16 hams (four of each gender and breed) were selected and analysed for volatile composition, using a dynamic headspace gas chromatography-mass spectrometry (DHS-GC-MS) technique. Seventy five compounds belonging to eight different chemical groups were identified in the hams. Alcohols were the most abundant constituents (41.38%) followed by aldehydes (31.53%). The possibility of classifying hams according breed and gender, using volatile compounds as independent variables, was analysed by principal component analysis, alcohols were the most suitable compounds to discriminate hams. Data from a larger number of samples are required to validate the conclusions.

Effects of including silage in the diet on volatile compound profiles and their modification during ripening of Montasio cheeses were examined. Twelve farms were selected and grouped according the type of forage in the ration: hay-based diets (four farms); hay and corn silage-based diets (four farms); and diets based on hay, corn silage and grass silages (four farms). For cheesemaking, 1000 kg of milk collected from two consecutive milkings was sampled from each farm and processed in the same cheese factory. Cheeses were ripened in the same cellar, in controlled humidity (78-85% relative humidity) and temperature (9-12 degrees C), until analysis. After 68 (62-74), 200 (194-206) and 360 (354-366) d of ripening, a cheese from each batch was analysed for moisture, protein and fat, volatile fatty acids (VFA) and volatile compounds. These latter were analysed by dynamic reverse carrier gas headspace gas chromatographic mass spectrometry (GC-MS) technique, scanning from m/z 29 to m/z 300 at 0.5 s cycle time. Total and individual VFA contents of cheeses did not differ between the different dietary treatments and increased linearly (P<0.01) during ripening, indicating that there were no appreciable defects of fermentation. Sixty-two volatile compounds were identified in the cheese samples: 12 aldehydes, 9 ketones, 16 alcohols, 17 esters, 2 hydrocarbons, 4 sulphur-containing compounds and 2 terpenes. Diet significantly affected the amount of total alcohols, ethanol, isobutanol, 1-penten-3-ol, 2-methyl-1-butanol and the sum of all the volatile compounds. Significant variations of 33 volatile compounds (9 aldehydes, 4 ketones, 6 alcohols, 10 esters, 1 hydrocarbons, 2 sulphur containing compounds, and 1 terpene) were observed during ripening. These results suggest that the influence of diet composition on volatile compounds of matured cheeses are related more to effects on microbial and chemical fermentations in cheese during ripening rather than to a direct transfer of molecules from milk.

Cell proliferation and apoptosis were measured in bovine mammary tissue around the time of peak milk production in three heifers, and compared with changes in the expression of bcl-2-related intracellular signals and apoptosis-inducing factor (AIF). Cell proliferation and apoptosis were relatively constant during the study period with no significant change in the incidence of either event. However, the ratio of apoptotic to proliferating cells tended to change from 0.99 on day 45 of lactation to 1.82 on day 63 (P=0.064), suggesting that in the course of the study, a dynamic balance may have been succeeded by net cell loss. Average milk production recorded 6 d after biopsy was correlated with the estimated number of cells (r=0.898; P<0.01) but not with the apoptosis to proliferation ratio (r=-0.224, P>0.05). Turnover of the cell population was associated with relatively constant expression of anti-apoptotic bcl-2. Competitive PCR also indicated expression of bax, in contrast to observations in lactating rodent mammary tissue. Bax expression was relatively low compared with that of bcl-2, but immunohistochemical staining for bax protein, which was not detectable on day 45, was observed on day 53 and, more intensely, on day 60 when the protein appeared to be membrane-associated. A partial coding sequence for bovine AIF was identified and AIF expression was evaluated by in situ hybridization. The results indicated that AIF was expressed in luminal alveolar cells and that, in concert with a change in bax to bcl-2 ratio, they might contribute to signalling of a change in the dynamic balance of the cell population as lactation progresses.

The composition of the volatile fraction of milk from cows was investigated in a survey of milk samples using a headspace sampling technique and gas chromatography coupled to mass spectrometry analysis (GC-MS). Milk samples were collected from 12 farms, selected for similar management, breed and level of production. Farms were also grouped according to the type of forage in the ration: (1) hay; (2) hay and maize silage; (3) hay, maize silage and grass silages. Forty-one compounds in milk were isolated and identified from GC-MS headspace analysis. Quantitatively, the most representative chemical class was ketones (eight compounds, 170 microg/kg), followed by aldehydes (nine compounds, 63 microg/kg), alcohols (eight compounds, 36 microg/kg), and lower amounts of hydrocarbons (six compounds), sulphur compounds (three compounds), esters (four compounds) and terpenes (three compounds). The novel headspace sampling technique, and the consequent reduction of sample pre-treatment, allowed the identification of low-molecular weight volatile compounds, and reduced the risk of producing artefacts during analysis. Discriminant analysis was used to identify a classification criterion for milk samples, using type of forage in the ration as a grouping variable. Posterior probability error rate indicated that aldehydes provided one of the best discriminant criteria for grouping milks according to ration composition. When all 41 identified volatile compounds were included, discriminant analysis selected nine compounds (acetone, 2,3-butanedione, 2-butanone, ethanol, acetaldehyde, ethylacetate, ethvlisovalerate, dimethylsulphone) that did not fail the tolerance test and which correctly classified 100% of the original cases.