Combinatorial Discriminant Analysis Applied to RNAseq Data Reveals a Set of 10 Transcripts as Signatures of Exposure of Cattle to Mycobacterium avium subsp. paratuberculosis

Paratuberculosis or Johne's disease in cattle is a chronic granulomatous gastroenteritis caused by infection with Mycobacterium avium subspecies paratuberculosis (MAP). Paratuberculosis is not treatable; therefore, the early identification and isolation of infected animals is a key point to reduce its incidence. In this paper, we analyse RNAseq experimental data of 5 ELISA-negative cattle exposed to MAP in a positive herd, compared to 5 negative-unexposed controls. The purpose was to find a small set of differentially expressed genes able to discriminate between exposed animals in a preclinical phase from non-exposed controls. Our results identified 10 transcripts that differentiate between ELISA-negative, clinically healthy, and exposed animals belonging to paratuberculosis-positive herds and negative-unexposed animals. Of the 10 transcripts, five (TRPV4, RIC8B, IL5RA, ERF, CDC40) showed significant differential expression between the three groups while the remaining 5 (RDM1, EPHX1, STAU1, TLE1, ASB8) did not show a significant difference in at least one of the pairwise comparisons. When tested in a larger cohort, these findings may contribute to the development of a new diagnostic test for paratuberculosis based on a gene expression signature. Such a diagnostic tool could allow early interventions to reduce the risk of the infection spreading

Evaluation of the chemical-nutritional parameters and the aromatic profile of eggs obtained from laying hens fed with a dietary supplementation of extruded linseed

Hen eggs represent a traditional food with an excellent nutritive value due to the presence of highly digestible proteins, vitamins, minerals and lipids, such as polyunsaturated fatty acids (PUFAs). Lipid composition of hen eggs is a subject of primary consumer concern, due to the relationship between specific dietary lipids and the development of coronary heart diseases (CHD). Nowadays, it is well known that ω-3 PUFAs provide important health benefits to humans as prevention and treatment of many ANIMAL PRODUCTS chronic diseases. The most significant ω-3 PUFAs appear to be α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and doco- sahexaenoic acid (DHA) and for the mentioned reasons over the course of time a growing interest has been developed in the pro- duction of eggs rich in ω-3 fatty acids, by feeding laying hens with experimental feedstuffs containing these nutrients. For example, ALA is predominantly in seed oils such as flax (Linum usitatissimum). Linseed is unique among oilseeds because of their exceptionally high content of ALA (≅50% of the total oilseed). The aim of this work was to determine the effect of the hens diet integration with extruded flaxseed (7%) on productive parame- ters and on the quality of resultant Bio-ω-3 eggs. At the end of the treatment, no significant difference was observed for eggs production while it was observed for the aver- age egg weight (58.05 ± 1.94% for control eggs vs. 63.37 ± 2.14% for flax eggs). Regarding, instead, the chemical-nutritional parameters, significant differences were not observed for total lipids and in the TBARs-test while significant differences were observed in the acidic profile. Specifically, ω-3 PUFAs were higher in flax eggs (p< .01) while SFA were higher in control eggs (p<.01). Also, β-carotene was found higher in flax eggs (478.20±15.19 μg/g vs. 324.80±13.84 μg/g, p<.001). The aro- matic profile was, also, analysed and significant differences were observed both for the alcohols and aldehydes compounds (p< .05). Finally, a significant difference in the colour was observed between the two types of eggs (ΔEab=1.77 ± 0.23, p<.05). In conclusion, it is possible to assert that the integration of the laying hens diet gave positive results as it has not negatively affected the production parameters, has improved the ω-6/ω–3 ratio (5.9:1 for flax eggs vs. 62:1 for control eggs) and the β-car- otene content, and has decreased the percentage of SFA guilty of cardiovascular pathologies

Treatment optimisation and sample preparation for the evaluation of lipid oxidation in various meats through TBARs assays before analysis

The aim of this work was to evaluate the critical points while using a TBARs test to measure the lipid oxidation in meat samples; this method includes a distillation method, extracting acid solutions (7% trichloroacetic acid—TCA, 4% perchloric acid—HClO4 and 4 N hydrochloric acid—HCl), butylated hydroxytoluene (BHT) concentrations and their interactions. The TBARs test method has been evaluated in different animal meat species and in different kinds of meat: fresh, stored, frozen and at different times of defrosting. Moreover, the influence of sample management was evaluated. The best results were obtained after using a distillation and a cold extraction with 7% TCA. The presence of the antioxidant agent (BHT) was essential and was more important in frozen samples than in fresh meat and especially when it was added at negligible concentrations immediately before defrosting, within 2 min of sample withdrawal from the freezer. The general management of the sample requires careful attention, avoiding storage and/or freezing, and lean meat appears to be more susceptible than fatty meat to the oxidative process

Effect of diet supplement in dairy cow with grape pomace on quality of milk and cheese

The aim of the study was to evaluate the effect of dairy cow diet supplement with grape pomace (GP) on quality of milk and cheese derived. The GP, a biomass deriving from winery and distillery industries, is a source of polyphenols and unsaturated fatty acids. Twelve lactating Holstein Friesan cows were randomly divided in two homogenous groups for parity, milk yield and days in milk. Cows were fed during 60 days with conventional ration in the control group (CG) and with GP (10% on daily dry matter intake) as supplement of conventional ration in the experimental one (EG). Samples of both diet were collected for chemical analyses. Milk production was monitored during the trial and individual milk samples were collected at the end of the experiment for both groups. Milk samples were analyzed to evaluate quality parameters (protein, casein, lactose, lipids, fatty acids profile, urea and somatic cells). Milk produced the last day of the experimental period was collected for both groups and used to produce cheese. Cheese was sampled at 1 (T1) and 30 (T30) days over ripening and fatty acids profile and lipid oxidation (TBARs test) were assessed. Statistical analysis was carried out using GLM procedure of SAS. Chemical analyses performed on diets highlighted a higher content of linoleic acid in EG diet compared with the CG one (46.73 vs 40.11%, respectively). Milk yield did not differ between two groups during the experimental time (16.58 vs 15.29 kg/d, CG and EG group respectively). Vaccenic (0.95 vs 1.45%), rumenic (0.55 vs 0.91%) and linoleic (2.05 vs 2.60%) acids were significantly higher in EG milk compared to CG one. Fatty acids composition of cheese, in particular vaccenic, rumenic and linoleic acids, were higher in EG group. Malondialdehyde did not differ at T1 between groups while it resulted almost three times lower in EG cheese after 30 days of ripening (0.117 vs 0.043 μg MDA/g). Results indicate that GP diet supplement in dairy cows may enhance milk and cheese fatty acids composition. The lowest lipid oxidation observed in treated cheese may be related to antioxidant properties of the higher concentration of polyphenols in grape pomac

TRANSCRIPTOMIC SIGNATURE OF HIGH DIETARY ORGANIC SELENIUM SUPPLEMENTATION IN SHEEP: A NUTRIGENOMIC INSIGHT USING A CUSTOM MICROARRAY PLATFORM AND GENE SET ENRICHMENT ANALYSIS

The objective of this study was to investigate the effect of a high dietary selenium (Se) supplementation on the whole-transcriptome of sheep. A custom-sheep whole-transcriptome microarray, with more than 23,000 unique transcripts, was designed, and then used to profile the global gene expression of sheep after feeding a high dietary supplementation of organic Se. Lactating cross-bred ewes (N = 10, 3 to 4 y of age; 55 to 65 kg BW) at late lactation [100 ± 8 d in milk (DIM)] were acclimated to indoor individual pen feeding of a basal control diet (0.40 mg Se/d, Na-selenite) for 4 wk. Sheep were then kept on a diet with an extra (high) supplementation of organic Se (1.45 mg Se/d as Sel-Plex, Alltech, Australia) for 40 d. Whole blood was collected at 2 time-points [last day of the acclimatization period (T0), and after 40 d of the organic Se supplementation (T40)], then total RNA was isolated and labeled for the subsequent microarray analysis. Significant analysis of microarray (SAM), using t-statistic, of the microarray data (T40 versus T0) evidenced the up- and down-regulation of 942 and 244 transcripts (false discovery rate; FDR < 0.05), respectively. Seven genes showed the same trend of expression (up- or down-regulation) when tested by qPCR in a cross-validation step. The microarray showed significant up-regulation of the following selenoproteins at T40: selenium binding protein 1 (SELENBP1), selenoprotein W1 (SEPW1), glutathione peroxidase 3 (GPX3), and septin 8 (SEPT8), and the expression trends for SEPW1 and SEPT8 were validated using qPCR. Functional annotation of the differentially expressed (DE) genes showed the enrichment of several immune system-related biological processes (lymphocyte activation, cytokine binding, leukocyte activation, T cell differentiation, B cell activation) and pathways (cytokine and interleukin signaling). Moreover, gene set enrichment analysis (GSEA) evidenced the enrichment of B and T cell receptors signaling pathways with an enrichment score (ES) of 0.63 and 0.59, respectively. Overall, from a global gene expression (whole-transcriptome) point of view, short-term supplementation of a high dietary organic Se to Se-nondeficient sheep results in a transcriptomic signature that mainly reflects an induced immune-system and a modulation of transcription effect. Also, the present study provides a custom whole-transcriptome microarray platform that can be used in further global gene expression studies in the ovine species

Effect of cow feeding supplementation with olive pomace on the development of aromatic compounds in milk and dairy products

Utilization of agro-industrial by-product in animal feeding represents an interesting and sustainable alternative to its disposal. The aim of this study was to evaluate the effect of olive pomace (OP) as supplement feed in dairy cow on aromatic compounds in raw milk, spontaneous fermented milk and cheese. Twenty lactating cows were randomly divided in two homogenous groups and fed for 60 days with OP supplement (2 Kg DM/cow/die) in the experimental group (EG) and without in the control one (CG). Diets were isoenergetic and isoproteic. Samples of bulk milk were collected at the end of the trial for both groups. Some of these were spontaneously fermented at 25 C for 72h and thereafter analyzed. The rest of bulk milk was used to produce cheese that was sampled at 1, 7 and 30 days of ripening. A SPME-GC-MS analysis was performed to detect volatile compounds. Data were analyzed with GLM procedure and significance were set at p<.05. In raw milk, besides free fatty acids, several secondary lipolysis catabolites (ethyl and methyl esters, lactones, aldehydes, methyl ketones) were detected and most of them (65%) were higher in EG (p<.01). Higher lypolitic activity (48% of compounds) in fermented milk was recorded in EG confirming that already observed in raw milk (p<.05). Esters deriving from methionine catabolism were higher in EG fermented milk (p<.05). On the other hand, data generally showed an increase of first catabolites of branched amino acids in CG milk, while 45% of their related esters were most detected in the EG (p<.05). Analysis of cheese highlighted differences (p<.05) between groups in some flavoring catabolites achieved at different times during ripening. Lypolitic catabolites were generally higher in the EG, especially at T7, with the exception for d-nonalactone that was most observed in CG at every time (p<.05). First leucine catabolites (a-ketoisocaproic acid, a-hydroxyisocaproic derivative) were greater in EG cheese at every time (p<.05), while 3-methylbutanol were higher in EG at T7 (p<.001) and T30 (p<.05). Their related esters, isoamyl isopentanoate and isoamyl butyrate, increased in EG at T7 and T30 (p<.01) and at T30 (p<.05), respectively. In conclusion, dietary supplement with olive pomace may affect lypolisis and proteolysis processes responsible of dairy products aroma. This pattern was already observed in raw milk and confirmed also in fermented milk and cheese, especially for compounds produced by esterase activity

High temperature and heating effect on the oxidative stability of dietary cholesterol in different real food systems arising from eggs

Cholesterol, a monounsaturated sterol present in food of animal origin, can be oxidized during food processing and storage, thus generating a series of oxidation products, collectively called oxysterols. These compounds are increasingly considered of potential interest in the onset and development of vascular disease due to their ability to trigger irreversible damage of vascular cells with consequent activation of phagocytes, up-regulation of the expression and synthesis of adhesion molecules and inflammatory cytokines. The understanding of the conditions, in the presence of which the cholesterol oxidation occurs in foods, could certainly help to prevent the accumulation of oxysterols, allowing to avoid harmful effects on human health. The aim of this work was to verify the presence of 7-ketocholesterol, the most representative of oxysterols family, in eggs and derivative obtained through different procedures of cooking. In the light of our results, we confirmed that the temperature and the lipid nature of the food matrix are closely related to the oxidative stability of cholesterol in foodstuffs. In particular, we tested 7-ketocholesterol formation in the presence of palm oil, rich in saturated fatty acids, or soybean oil, rich in polyunsaturated compounds

RNA sequencing-based transcriptome profiling of dairy cows fed with a polyphenol-rich grape pomace-supplemented diet

The inclusion of some agro-industrial by-products in animals’ diet is becoming attractive not only for being part of an ambitious waste management and sustainability policies but also due to its possible nutritive values. The aim of this study was to evaluate the effect of grape pomace (GP) - the polyphenolrich by-product of the wine industry – on the transcriptome of dairy cows. Twelve lactating Holstein Friesian cows, homogeneous for age and lactation period, were assigned to two groups of six animals each - in a randomized pretest-posttest control group design. The first group received a basal diet and served as a control (CTR), while the other received a 10% GP-supplemented diet for 67 days. Whole blood was collected from each group at 2 time-points [beginning (T0), and after 67 days of the GP supplementation (Tf)], then total RNA was isolated, quality-controlled, and then used for library preparation. The sequencing of twenty-four samples (2 groups6 animals/group2 time-points) resulted in an average of 17 million reads per sample. The 50bp single-end reads were quality-controlled, mapped to the Bos taurus reference genome (UMD 3.1 assembly), then tested for the presence of differentially expressed genes (DEGs) in the same group (CTR or GP) after (compared with before) 67-days-supplementation period (Tf vs T0). On average, 95.6% of the reads were mapped to the reference genome. Reads mapped to exons were counted with HTSeq-count, then analysed by the DESeq2 R package. The bioinformatics analysis evidenced a significant (adjusted p<.1) change in expression of 14 and 88 genes in the CTR and GP groups, respectively. Four genes were found to be overlapping between the two groups, thus they were excluded from the GP group results as being ‘temporally’- and not experimentally-affected DEGs. Of the remaining 84 GP-affected genes, 73 were down-regulated, with most of them being ‘ribosomal protein’-coding genes. The functional analysis evidenced the positive enrichment of ‘defence response to other organism’ (p¼.0002) biological process and the ‘interleukin signalling’ pathway (p¼.0002), as well a negative enrichment of the ‘ribosome’ pathway (35 genes, p¼5x1055). Overall, the transcriptomic signature of GP-supplemented diet reflects an induced immune system and a suppressed ‘ribosome biogenesis’, which can b

Characterization of the rumen microbiota in dairy calves receiving copper or grape-pomace feed supplementatio

In this study, the rumen microbiota of Holstein-Friesian calves was characterized, and the effect of two feed supplementations on its composition was investigated. Fifteen male calves of an average age of 195 days were assigned to three groups: copper supplementation (cupric sulphate: 300 g/100L of drinking water), grape pomace supplementation (1 kg/head/ day), and control (no supplementation). The dietary treatment had a duration of 75 days. Copper is an essential trace elements; grape pomace is a source of polyphenols and resveratrol which have antioxidant and protective properties. Rumen fluid was sampled after slaughter for microbial-16S metabarcoding- sequencing. Taxonomic counts were then used to characterize the rumen microbiota and assess the impact of dietary supplementation. The average number of different microbial OTUs (operational taxonomic units) was highest in the grape-pomace group (1583) compared to the copper (1507) and control (1500) groups. The species richness indicators (Chao1, abundancebased coverage estimator-ACE) gave a similar picture. The Shannon and Simpson diversity indexes were very similar among groups. On the contrary, the Fisher’s alpha index showed a tendency of being higher in grape pomace group (256.4) than in copper and control ones (235.1 and 218.6 respectively). Overall, the most abundant microbial species in the rumen were Prevotella stercorea, Bacteroides uniformis and Ruminococcus flavefaciens, which accounted for 79% of total rumen bacteria. The same three species were the most abundant in the grape-pomace group (86% of the total), in controls (76% of total) and in the copper group (70% of total). Microbial counts were also used to classify calves in the three experimental groups. Random Forest (RF) was used for classification. When all OTUs were fitted to the classification model, the OOB (out-of-bag) classification error was high (66%). However, if only the four most relevant OTUs (from RF variable importance ranking) were fitted to the RF model, the OOB error dropped to 26.7%. The rumen microbiota in dairy calves seems to be dominated by few taxas. Feed supplementation with grape-pomace and copper did not have a profound impact on the overall composition of the rumen microbiota. However, specific microbial species and/or taxa were differentially abundant in the treatment groups, and allowed for a certain degree of discrimination between them