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AbstractA growing number of studies in the last decade described the microbial populations in different niches of the organism thank to High-throughput DNA sequencing techniques, that are easily accessible to researchers. Furthermore, network analysis allows characterisation of bacteria that are indirectly linked to outcomes such as disease, diet and host sex through their association with other taxa. The present work on the gut microbiome follows previous research in which data were collected from 8 in-house dietary intervention studies in healthy dogs. Animals were divided according to diet and sex. The process of sample collection, storage, DNA extraction and sequencing, bioinformatic and statistical analysis followed a tailored, internally defined pipeline. The extracted DNA was prepared for sequencing of the V3 and V4 regions of the 16 rRNA gene. Correlation network analysis was performed by calculating pairwise relationships between taxa using the Sparse Correlations for Compositional Data (SparCC) algorithm. First, we identified candidate bacteria that were highly abundant in the microbial community, and second, we examined taxa that were directly related to diet and sex factors. In summary, this study underpins the network structure of the gut microbiome of dogs categorised by diet and sex and provides a better explanation for the interactions between bacteria that led to the grouping of dogs based on environmental or genetic factors. Specifically, this study has provided the basis for understanding how the bacterial community in the gut is interconnected and functions as a function of diet composition and sex.HighlightsNetwork analysis could allow indirect association between bacteria composing the gut microbiome and outcomes, such as disease, diet and sex of the host.This research put the basis on the definition of enterotypes also in dogs, a concept already applied in humans.Positively correlated bacteria, belonging to modules could help to modulate diets and therapies for the treatment of enteropathies or gut-related pathologies linked to a dysbiosis status of the host.

AbstractMetabolomics provides a description of the phenotype of an organism and complementary biochemical information to genomics and proteomics. The purpose of this research was to depict the metabolite profile of faecal samples from dogs fed three different diets through NMR spectroscopy analysis. Samples were collected from 14 dogs fed a commercial extruded diet, 18 dogs fed a homemade diet and 16 dogs fed a raw meat-based diet. The average BCS for all dogs was 4–5 and the average Faecal Score was 2–3. Only healthy animals were considered, as assessed from the clinical evaluation of the veterinarians. Faecal samples were prepared using phosphate buffer (pH 7.1) combined with deuterated water and analysed with NMR spectroscopy using a Bruker Avance III HD 400 MHz spectrometer. Principal component analysis of the spectra signals demonstrated clustering of dogs according to diet, with 57.8% of the variance explained by the first three components. Targeted metabolome analysis was also performed on 56 metabolites of interest, selected from a database of 558 metabolites. Our data suggest that metabolome analysis using NMR is a promising approach to describe the phenotypic variation that occurs among dogs fed different diets.The metabolomic approach is becoming always more important in the evaluation of the functional responses of animals to dietary intervention.NMR-metabolomics has emerged as a non-destructive technique that allows a simpler data interpretation compared to other techniques.NMR metabolome analysis resulted to be a promising approach to describe the phenotypic variation that occurs among dogs fed different diets.

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–NH3) 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–NH3and 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 SMB53genus at the end of the study in B1 diet and of Megamonasgenus 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.

AbstractThe aim of the study was to investigate if the inclusion of chickpeas or peas in the diet can modify faecal microbiome in dogs. Eight healthy adult Border collie, fed a commercial extruded diet as reference diet (RD), were divided in two groups of four individuals. At the beginning of the trial, one group received a diet based mainly of raw meat, rice and chickpeas (CP) and in the other group this pulse was substituted with peas (PE). After 14 days, the dogs with CP diet shifted to the PE and those with PE shifted to the CP diet, for another 14 days. Faeces were collected at the beginning (T0), after 14 days (T14) and at the end of the study (T28). Faeces were analysed for 16S rRNA, short chain fatty acids (SCFA), lactate, pH and faecal score was also evaluated. The SCFA and lactate in the faeces were not affected by the inclusion of pulses, with the only exception of isovalerate, which was higher in CP and PE diets in comparison with RD diet (p< .05). The abundances of Erysipelotrichaceae incertae sedis, Eubacterium, Anaerobacterand Sarcinasignificantly differed in CP and PE in comparison with RD. Moreover, the genera Prevotella, Lactobacillus, Alloprevotella, Suttarellavaried significantly between CP and PE diets. The observed modifications of faecal microbioma were related not only to the change from RD to CP or PE, but also to the type of pulse, chickpeas or peas. However, long-term studies are required to investigate the implications that pulses can have for gut health.HighlightsFaecal quality and end products of fermentation were similar between diets.Faecal microbiota is affected by the type of pulse and raw meat inclusion.Thermally treated Chickpeas or Peas in the diet of dogs were safe, but further study are required.

AbstractThe aim of the study was to investigate element concentrations in the hair of growing and adult dogs. Overall, 39 dogs were recruited in a kennel, split in 4 groups: G1, 10 puppies of small size breeds; G2, 6 puppies of medium size breed; G3, 11 adult dogs of medium size breeds; G4, 12 adult dogs of toy breeds. The dogs of each group were fed 4 different complete diets, according to the requirements. Hairs were sampled at the beginning of the study (D0) and after 60 days (D60). Elements were analysed in triplicate using ICP-OES. In the hair of the adult dogs of the G3 group the concentration of Al (p < .01) was lower and in the G2 group the concentration of Zn was higher (p < .01) than in the other groups. Cu and Mn concentrations in the hair were higher (p < .01 and p < .05, respectively) in puppies fed G1 and G2 diets in comparison to adult dogs fed G3 and G43 diets. Hair concentrations of K, Li and Na were higher at D60 in comparison with D0 (p < .01). Correlations between concentrations in the hair of Al and Fe, Al and Mg and Fe and Mg were highly significant (p < .01). Present data do not support the use of element concentrations in the hair to assess their nutritional supply but, likely, they can be used in relation to physiological status of the animals. Further studies are required to investigate the factors affecting element concentrations in the hair of dogs.HighlightsConcentrations of Cu, Mn and Zn in the hair differed between growing and adult dogs;Al concentration in hair was highly correlated with Fe and Mg;Several factors other than nutritional supply regulate element deposition in the hair

AbstractWorking dogs are gaining popularity for their ability to learn and perform tasks entertaining their human companions. For this reason, dogs are often subjected to various stimuli due to inter- and intra-specific interactions, environmental variations and effort required by different activities. In the present study, salivary cortisol was measured to monitor physiological response to different conditions. The first study was performed to assess the variability of salivary cortisol in dogs in usual environmental conditions. For this, salivary cortisol was measured in 10 dogs at home during three not consecutive days at three different times of the day and not significant variations between days and time of sampling were observed. In the second study, salivary cortisol was measured in dogs before and after Pointing Hunting (No. 5), Tracking for Ungulate Hunting (No. 6), Blood Tracking (No. 4), Agility Training (No. 6) and Animal Assisted Activities (AAA, No. 6). Salivary cortisol concentration significantly increased after the Pointing Hunting activity (p < .05), while salivary cortisol significantly decreased at the end of AAA session (p < .05). No significant differences in cortisol variations were observed for Tracking for Ungulate Hunting, Blood Tracking and Agility Training, before and after the activities. The response of cortisol suggests that the extent of hypothalamus–pituitary–adrenal axis activation varies between short high-intensity activities and endurance exercises. The measurement of salivary cortisol can support the trainers to evaluate the animal response to the stimulations.

AbstractThe study aimed at investigating the rumen microbiome composition and functional activity in mid lactating cows of Italian Holstein (IH) and Italian Simmental (IS) breeds. Eight IH and eight IS pluriparous cows with days in milking (DIM) ranging from 90 to 180 were selected and rumen contents were sampled with oesophageal tube. Rumen metagenome was analysed using a whole shotgun sequencing. Data were analysed for taxonomic classification and microbial genes. The relative abundance of Archaea, the Archaea to Bacteria ratio and the Archaea to Eukarya ratio were higher (p < .05) in IS than IH cows. The comparison between IH and IS underlined differences for the abundances of Bacteria, being Bactroidaceae, Bacteroides, Prevotellaceaeand Prevotellalower (p < .05) in IS than in IH cows. The IS cows showed higher abundances of Euryarchaeota(p < .05), Methanosphera(p < .01) and Methanothermobacter(p < .05) than IH cows. The annotation of sequences to KEGG revealed that 170 genes were differentially abundant between IS and IH cows and among these, 20% were involved in protein biosynthesis, 8.8% in one-carbon metabolism, as methyl coenzyme M reductase associated protein and of six isoforms of methyl coenzyme M reductase. The present results suggest a genetic link between breed and microbiome, although this interaction can be influenced by several biological factors. Considering that there are still a low number of whole genome shotgun sequencing analysis of rumen communities, these data can provide further information to scientific community.

AbstractRuminant production has been an essential part of human activities worldwide since ancient times. The expected increase in world population and per capita income, with an increase in the amount and prevalence of animal products in human diet, urbanisation, with a concentration of population in urban areas and an increase in losses in the supply chain, and the growing concern over the environmental impact of animal farming require a long-term global strategy for a more intensive and sustainable ruminant production. Therefore, solutions to increase the supply of high-quality products of ruminant origin, without harming human health, animal welfare, and environment, should consider the following interconnected issues discussed in this review: (a) effects of meat, milk and dairy products consumption on human health, focussing on the imbalance caused by their insufficient consumption, and the alleged increased incidence of certain diseases due to their consumption; (b) importance of the sustainable intensification of ruminant production systems (e.g. better feed conversion and higher production output per unit of input introduced into the farming system); (c) environmental impact of ruminant production; (d) improvement of animal performance by improving animal welfare; (e) adaptation of ruminants to climate change; (f) sustainable ruminant feeding (e.g. precision feeding techniques, optimisation of grazing systems, and use of unconventional feeds); (g) challenges posed by production intensification to animal breeding and conservation of animal biodiversity; and (h) strategies to increase ruminant production in developing countries, thus achieving food security in vast areas of the planet affected by fast growth of human population.

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 DLSCCand 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 DMEOwere characterized by a lower content of LSCC and DSCC, and higher DLSCCand DLDSCC, compared to the low DMEOherds. 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 DLDSCCcompared 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 results obtained from animal-related factors stimulate the investigation of biological pathways and genetic background of total and differential somatic cell traits. Moreover, these findings address the opportunity of using the information of milk production when setting thresholds based on somatic cells for identifying udder inflammation. Hence, it could be possible to set new thresholds related to the number of somatic cells produced per day, in order to help farmers properly manage their cows and herds.This study investigated factors affecting the variability of somatic cell traits in bovine milk. Animal had greater influence on somatic cell score (SCS) and differential somatic cell count (DSCC) compared to herd factors. Herds producing high average of daily milk energy were characterized by lower SCS and DSCC compared to the low average daily milk energy herds. The SCS and DSCC were higher in Holstein-Friesian than in Simmental, and during summer with respect to the other seasons. Older cows at the end of lactation showed the highest content of somatic cell traits. These results are helpful for the management of somatic cell traits at herd and animal levels.

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°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&lt;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&lt;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 obese gene encodes leptin, a 16-kDa protein involved in the regulation of fat deposition and energy consumption. Backfat is one of the peculiar characteristics of Italian ham, and represents a fundamental quality factor. Therefore, the obese gene can be considered as a candidate marker for determining economically important production traits such as backfat thickness, feed intake, and growth rate in swine. The aim was to investigate the relationship between obese gene polymorphisms and carcass and meat characteristics of pigs reared for ham production. In the present research, the analyses of three new RFLPs are reported. An MwoIpolymorphism occurs at nucleotide 1792, within the intron. Pigs heterozygous at this position have heavier thighs with a thinner layer of fat. Two SmaI polymorphisms occur at nucleotides 5018 and 5410 within the 3́ UTR of the obese gene. Animals heterozygous at position 5410 have characteristics suitablefor the production of San Daniele ham: lower backfat thickness and heavier thighs with a thinner fat layer, relative to other genotypes

The decline in milk yield after peak lactation in dairy animals has long been a biological conundrum for the mammary biologist, as well as a cause of considerable lost income for the dairy farmer. Recent advances in understanding the control of the mammary cell population now offer new insights on the former, and a potential means of alleviating the latter. The weight of evidence now indicates that a change in mammary cell number, the result of an imbalance between cell proliferation and cell removal, is a principal cause of declining production. Further, it suggests that the persistency of lactation, the rate of decline in milk yield with stage of lactation, is strongly influenced by the rate of cell death by apoptosis in the lactating gland. Mammary apoptosis was first demonstrated during tissue involution after lactation, but has now been detected during lactation, in mammary tissue of lactating mice, goats and cattle. Those factors that determine the rate of cell death by apoptosis are as yet poorly characterized, but include the frequency of milking in lactating goats. Initial evidence suggests that nutrition also is likely to influence cell survival after peak lactation, an important factor being the degree of oxidative stress imposed by feed and the tissue's ability to deal with, and prevent damage by, reactive oxygen species. Comparison of cows in calf or not pregnant during declining lactation also indicates a likely influence of reproductive hormones, with oestradiol and progesterone acting to preserve mammary ductal and alveolar integrity during the dry period, while allowing a degree of apoptosis and cell replacement. In each case, the molecular mechanisms controlling mammary cell survival (or otherwise) are as yet poorly defined. On the other hand, more persistent lactations are likely to benefit animal welfare through fewer calvings and by placing less emphasis on maximal production at peak lactation, and modelling of persistent lactation with longer calving intervals indicates their likely economic benefits. In these circumstances, there is considerable incentive to elucidate the determinants of mammary apoptosis, and the factors controlling the dynamic balance between cell proliferation and cell death in the lactating mammary gland.

AbstractThe effect of Larix decidua(LD) dietary administration on gene expression patterns has been evaluated in sheep under ACTH challenge. Experimental protocol was approved by local laws and regulations. Eighteen sheep at maintenance were allotted to 3 groups: CTR (negative control, without ACTH, and supplementation), ACTH (positive control, with ACTH, and without supplementation), LD (ACTH and 50 g/head/day of LD). ACTH was injected for 3 subsequent days to ACTH and LD groups and blood was sampled before (T0) and after 3 (T3) and 51 (T51) hours from the first injection. RNA extracted samples were pooled together within group and time of sampling. A custom oligoarray was synthesized using 24,384 35 -40mer probes designed from 12,194 UniGenes (NCBI) on a CombiMatrix 90K platform. Cy5 labelled samples were hybridized on the chip. Statistical analysis, performed with MeV software 4.1 (TIGR), allowed the identification of a set of genes which were up or down regulated as a consequence of ACTH treatment. Genes that resulted differentially expressed were annotated with HomoloGene system and data mining was performed with Babelomics v3.1 tool. Functional analysis showed that most of the differentially expressed genes belong to KEGG pathways involved in immune system response and signaling molecules and interaction. Larch administration was effective in counteracting the effect of ACTH injection on the inflammatory processes, restoring the physiological homeostasis.

AbstractPro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor- alpha (TNF-α) play a key role in the pathogenesis of osteoarthritis (OA). Once released, these cytokines are potent stimulators for the de novo production of catabolic enzymes such as matrix metalloproteinases (MMPs) and cyclo-oxygenase-2 (COX-2). Anti-inflammatory agents capable of suppressing the production and catabolic actions of these cytokines may have therapeutic potential in the treatment of OA and a range of other osteoarticular disorders. The purpose of this study was to examine the therapeutic effect of Curcumin (diferuloylmethane), a pharmacologically safe phytochemical agent, on males and foals affected by degenerative joint diseases. Curcumin, in the form phytosome (CURCUVET®, Indena Spa, Milan, Italy) was administered to animals for fifteen days and gene expression was monitored before the treatment and after four, eight, and fifteen days. In mares, Curcumin inhibited the expression of COX-2, TNF-α, IL-1β, IL1RN, and IL6, even if only the downregulation of IL-1β and IL1RN were significant. In foals, Curcumin significantly inhibited the expression of COX-2, TNF-α, IL1RN and significantly increased that of IL6. These results indicate that Curcumin has nutritional potential as a natural anti-inflammatory agent for treating osteoarticular disorders through suppression of pro-inflammatory cytokines and catabolic enzymes.

The study investigated the relationship of markers of welfare and metabolism in milk, urine and blood with the genetic merit of Holstein and Simmental cows after the peak of lactation. Cows were selected from 3 Simmental (IS) and 2 Holstein (IH) commercial dairy farms. Within each farm, cows were ranked according to the estimated breeding value for milk protein yield (EBVp) from minus to positive and selected every 5 EBVps from minus to positive values (about 20% lactating cows for each farm). Milk was sampled and analysed for protein, fat, lactose, cortisol contents and somatic cell count (SCC). Blood and urines were analysed for biomarkers of metabolism and welfare. Significantly lower body condition score (BCS) was observed for IH in comparison to IS. Plasma creatinine was higher in IS, whilst Zn, total antioxidant status and glutathione peroxidase was higher in IH. The creatinine N to N ratio in urine was significantly higher for IS, while the purine derivatives (PD) N to creatinine N ratio was higher for IH. The EBVp was negatively related to BCS and glucose for IS and to plasma b-hydroxybutyrate in both breeds. EBVp was negatively related to urinary PD N to total N ratios for IS and to PD N to creatinine N ratio for IH. These preliminary results would indicate that the selection of cows for milk protein yield had minor effect on plasma and milk biomarkers of welfare. Instead, biomarkers of metabolism were more affected by breed and genetic merit.