Inulin-induced differences on serum extracellular vesicles derived miRNAs in dairy cows suffering from subclinical mastitis.

MicroRNA (miRNA) profiles vary with the nutritional and pathological conditions of cattle. In this study, we aimed to investigate the effects of inulin supplement on miRNA profiles derived from serum extracellular vesicles (EVs). Our goal was to determine the differences in miRNA expressions and analyse the pathways in which they are involved. Based on the results of California mastitis test and milk somatic cell counts, ten lactating cows with subclinical mastitis were randomly divided into two groups: an inulin group and a control group (n = 5 in each group). The inulin group received a daily supplement of 300 g of inulin while the control group did not receive any supplementation. After a 5-week treatment period, serum-derived EV-miRNAs from each cow were isolated. High-throughput sequencing was conducted to identify differentially expressed miRNAs. GO and KEGG bioinformatics analysis was performed to examine the target genes of these differentially expressed miRNAs. The EV-RNA concentration and small RNA content were not affected by the inulin treatment. A total of 162 known miRNAs and 180 novel miRNAs were identified from 10 samples in the two groups. Among the known miRNAs, 23 miRNAs were found to be differentially expressed between the two groups, with 18 upregulated and five downregulated in the inulin group compared to the control group. Pathway analysis revealed the involvement of these differentially expressed miRNAs in the regulation of cell structure and function, lipid oxidation and metabolism, immunity and inflammation, as well as digestion and absorption of nutrients. Overall, our study provides a molecular-level explanation for the reported beneficial health effects of inulin supplementation in cows with subclinical mastitis.

Invited review: From heat stress to disease-Immune response and candidate genes involved in cattle thermotolerance.

Heat stress implies unfavorable effects on primary and functional traits in dairy cattle and, in consequence, on the profitability of the whole production system. The increasing number of days with extreme hot temperatures suggests that it is imperative to detect the heat stress status of animals based on adequate measures. However, confirming the heat stress status of an individual is still challenging, and, in consequence, the identification of novel heat stress biomarkers, including molecular biomarkers, remains a very relevant issue. Currently, it is known that heat stress seems to have unfavorable effects on immune system mechanisms, but this information is of limited use in the context of heat stress phenotyping. In addition, there is a lack of knowledge addressing the molecular mechanisms linking the relevant genes to the observed phenotype. In this review, we explored the potential molecular mechanisms explaining how heat stress affects the immune system and, therefore, increases the occurrence of immune-related diseases in cattle. In this regard, 2 relatively opposite hypotheses are under focus: the immunosuppressive action of cortisol, and the proinflammatory effect of heat stress. In both hypotheses, the modulation of the immune response during heat stress is highlighted. Moreover, it is possible to link candidate genes to these potential mechanisms. In this context, immune markers are very valuable indicators for the detection of heat stress in dairy cattle, broadening the portfolio of potential biomarkers for heat stress.

Effect of dietary protein and energy intake on embryonic survival and gene expression in the uterine endometrium of early pregnant gilts.

Porcine embryonic loss during early gestation is a serious problem in swine production. Improving embryonic survival can be achieved by maternal manipulation. Protein and energy are two major components of the diet, which play decisive roles in embryonic survival. This study was performed to evaluate the effects of enhancing maternal protein or energy intake on embryonic survival during early gestation in gilts and to explore the underlying mechanism. From day (d) 0 to 30 of gestation, 40 gilts (Landrace × York) were randomly allocated to 5 diets according to daily intake of low (L, National Research Council (NRC) recommendation for gestation gilts), medium (M, 20% higher than NRC) or high (H, 40% higher than NRC) CP or metabolisable energy (ME) (LCPLME, MCPLME, HCPLME, LCPHME, HCPHME). Gilts were sacrificed on d 30 of gestation, and number of foetuses and corpora lutea, embryonic survival rate, uterine weight, and total volume of allantoic fluid were recorded or calculated. Gene expression was determined by Quantitative Real-time PCR (qPCR), western blot or immunohistochemistry. Results showed that increasing protein or ME intake significantly increased embryonic survival rate. Compared with diet LCPLME, plasma progesterone (P4) concentration in diet LCPHME increased at d 14 and d 30 of gestation. Progesterone receptor (PGR) was found not to be expressed in the epithelia but was strongly expressed in the stroma of the endometrium. Increasing protein or ME intake did not alter PGR expression in the endometrium. There was also no change in the amount of P4, hepatocyte growth factor, and fibroblast growth factor-7 in the endometrium. The mRNA abundance of cationic amino acid transporter 1 in the endometrium in diet LCPHME and HCPHME was significantly lower than in diet LCPLME. Diet HCPLME showed a tendency to increase neutral amino acid transporter 1 mRNA expression in the endometrium compared to diet LCPLME (P = 0.087). In conclusion, increasing maternal protein or ME intake had a positive effect on the embryonic survival. Increased protein intake by 20 or 40% did not alter plasma P4 level, but increasing ME intake by 40% improved plasma P4 concentration at d 14 and 30 of gestation. Increasing maternal protein or ME intake did not induce PGR expression in the endometrium. Maternal protein and energy intake likely mediate transportation of cationic and neutral amino acids from mother to foetus to affect embryonic survival and development.

Interaction of CP levels in maternal and nursery diets, and its effect on performance, protein digestibility, and serum urea levels in piglets.

Reduced protein levels in nursery diets have been associated with a lower risk of postweaning diarrhea, but the interaction with CP levels in maternal diet on the performance of the offspring remains unclear. The objective of this study was to determine the effect of protein content in sow gestation and piglet nursery diets on the performance of the piglets until slaughter. This was studied in a 2 × 2 factorial trial (35 sows, 209 piglets), with higher or lower (H or L) dietary CP in sow diets (168 vs 122 g CP/kg) during late gestation. A standard lactation feed was provided for all sows (160 g CP/kg). For both sow treatments, half of the litters received a higher or lower CP in the piglet nursery diet (210 vs 166 g CP/kg). This resulted in four possible treatment combinations: HH, HL, LH and LL, with sow treatment as first and piglet treatment as second letter. For each phase, all diets were iso-energetic and had a similar level of essential amino acids. Ps*p is the p-value for the interaction effect between sow and piglet treatment. In the nursery phase (3.5-9 weeks of age), a tendency toward interaction between piglet and sow treatments with feed efficiency (Ps*p = 0.08) was observed with HH having the highest gain:feed ratio (G:F) (0.74 ± 0.01), LH the lowest (0.70 ± 0.01) and the other two groups intermediate. In the growing-finishing phase, an interaction was observed between the piglet and sow diets with decreased G:F for LH (Ps*p = 0.04) and a tendency toward interaction with increased daily feed intake for LH (Ps*p = 0.07). The sow diet showed a tendency toward a long-lasting effect on the dressing percentage and meat thickness of the offspring, which was higher for the progeny of H sows (Ps < 0.01 and Ps = 0.02, respectively). At 23 weeks, serum urea concentrations tended to be lower for the HH and LL groups (Ps*p = 0.07). Fecal consistency scores were higher at day 10-day 14 after weaning for piglets from L sows (Ps = 0.03 and Ps < 0.01, respectively). At day 7 after weaning, fecal consistency score was higher for piglets fed the higher protein diet (Pp < 0.01). At 8 weeks of age, the apparent total tract digestibility of CP (ATTDCP) interacted between piglet and sow diet (Ps*p = 0.02), with HH showing the highest digestibility values. In conclusion, the protein levels in sow late-gestation and piglet nursery diets interacted with feed efficiency, ATTDCP and serum urea concentrations in the nursery phase.

Individual differences in behaviour and gut bacteria are associated in collared peccary (Mammalia, Tayassuidae).

AIMS: We tested the hypothesis that the behaviour of an individual is associated with the diversity of its gut bacteria, using the collared peccary (Pecari tajacu) as a model. METHODS AND RESULTS: In all, 24 adult male collared peccaries received either low- (n = 12) or high-fibre diet (n = 12) to induce contrasting gut fermentation profiles. They were submitted to three short-term challenges, allowing us to rate the animals in a coping-style dimension named 'calmness'. At the end of the experimental period, we collected samples of peccaries' forestomach contents to characterize bacterial diversity. We found a significant positive association between individual 'calmness' z-scores and the bacterial evenness index in gut bacteria (and a similar trend with the Simpson's diversity index), suggesting a more homogeneous bacterial community of calmer individuals. We also found a positive association between fibres digestibility and gut bacterial diversity in the peccaries' forestomach, but no effect of the dietary fibre level. CONCLUSIONS: Gut bacteria evenness increases with 'calmness' z-scores, suggesting a more homogeneous bacterial community of calmer individuals, compared with the more heterogeneous of the most distressed ones. Our results also suggest associations between the digestibility of ADF with the gut bacterial diversity indices and with the relative abundance of the Actinobacteria phylum. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data showed that the hosts' individual behavioural differences are potentially aligned with gut bacterial diversity. The behaviour-microbiota link is correlated with host feed efficiency and, ultimately, may have implications for animal health and welfare of farm animals.

Direct and maternal reduced balanced protein diet influences the liver transcriptome in chickens.

The objective of this study was to evaluate, by means of RNA sequencing, the direct and transgenerational effect of a reduced balanced protein (RP) diet on broiler breeder metabolism. Chickens of the F0 generation were fed a control (C) or RP diet, and their F1 progeny was fed a C or RP diet as well, resulting in four groups of chickens: C/C, C/RP, RP/C and RP/RP. While both direct and maternal effects were seen on body weight, breast muscle weight and abdominal fat weight in the F1 generation, the direct effect was the most dominant one. The liver transcriptome in the F1 generation showed that amino acid metabolism was up-regulated in chickens that received the control feed when compared with their respective contemporaries that received the reduced protein diet. Interestingly, chickens hatched from control-fed hens but reared on the reduced protein diet (C/RP group) activated a fatty acid metabolism, expressing more fatty acid desaturase 1 gene, fatty acid desaturase 2 gene and elongation of very long-chain fatty acids protein 2 gene, when compared with control-fed chickens hatched from control-fed hens (C/C group), while chickens hatched from reduced protein-fed hens that received themselves the same reduced protein diet (RP/RP group) triggered their glucose metabolism more, showing elevated levels of phosphofructokinase gene, 6-phosphofructo-2-kinase/fructose-2,6-biphospatase 4 and fructose-biphosphate aldolase C mRNA compared with the chickens hatched from reduced protein-fed hens but reared on a control diet (RP/C group). This suggests that the maternal protein diet has an impact on the metabolism of broilers when they are reared on a RP diet.

Genomewide association of piglet responses to infection with one of two porcine reproductive and respiratory syndrome virus isolates.

Porcine reproductive and respiratory syndrome (PRRS) is a devastating disease in the swine industry. Identification of host genetic factors that enable selection for improved performance during PRRS virus (PRRSV) infection would reduce the impact of this disease on animal welfare and production efficiency. We conducted genomewide association study (GWAS) analyses of data from 13 trials of approximately 200 commercial crossbred nursery-age piglets that were experimentally infected with 1 of 2 type 2 isolates of PRRSV (NVSL 97-7985 [NVSL] and KS2006-72109 [KS06]). Phenotypes analyzed were viral load (VL) in blood during the first 21 d after infection (dpi) and weight gain (WG) from 0 to 42 dpi. We accounted for the previously identified QTL in the region on SSC4 in our models to increase power to identify additional regions. Many regions identified by single-SNP analyses were not identified using Bayes-B, but both analyses identified the same regions on SSC3 and SSC5 to be associated with VL in the KS06 trials and on SSC6 in the NVSL trials ( < 5 × 10); for WG, regions on SSC5 and SSC17 were associated in the NVSL trials ( < 3 × 10). No regions were identified with either method for WG in the KS06 trials. Except for the region on SSC4, which was associated with VL for both isolates (but only with WG for NVSL), identified regions did not overlap between the 2 PRRSV isolate data sets, despite high estimates of the genetic correlation between isolates for traits based on these data. We also identified genomic regions whose associations with VL or WG interacted with either PRRSV isolate or with genotype at the SSC4 QTL. Gene ontology (GO) annotation terms for genes located near moderately associated SNP ( < 0.003) were enriched for multiple immunologically (VL) and metabolism- (WG) related GO terms. The biological relevance of these regions suggests that, although it may increase the number of false positives, the use of single-SNP analyses and a relaxed threshold also increased the identification of true positives. In conclusion, although only the SSC4 QTL was associated with response to both PRRSV isolates, genes near associated SNP were enriched for the same GO terms across PRRSV isolates, suggesting that host responses to these 2 isolates are affected by the actions of many genes that function together in similar biological processes.

Transcript profiles in longissimus dorsi muscle and subcutaneous adipose tissue: a comparison of pigs with different postweaning growth rates.

Although most pigs recover rapidly from stresses associated with the transition of weaning, a portion of the population lags behind their contemporaries in growth performance. The underlying biological and molecular mechanisms involved in postweaning differences in growth performance are poorly understood. The objective of this experiment was to use transcriptional profiling of skeletal muscle and adipose tissue to develop a better understanding of the metabolic basis for poor weaned-pig transition. A total of 1,054 pigs was reared in commercial conditions and weighed at birth, weaning, and 3 wk postweaning. Transition ADG (tADG) was calculated as the ADG for the 3-wk period postweaning. Nine pigs from both the lowest 10th percentile (low tADG) and the 60th to 70th percentile (high tADG) were harvested at 3 wk postweaning. Differential expression analysis was conducted in longissimus dorsi muscle (LM) and subcutaneous adipose tissue using RNA-Seq methodology. In LM, 768 transcripts were differentially expressed (DE), 327 with higher expression in low tADG and 441 with higher expression in high tADG pigs (q < 0.10). Expression patterns measured in LM by RNA-Seq were verified in 30 of 32 transcripts using quantitative PCR. No DE transcripts were identified in adipose tissue. To identify biological functions potentially underlying the effects of tADG on skeletal muscle metabolism and physiology, functional annotation analysis of the DE transcripts was conducted using DAVID and Pathway Studio analytic tools. The group of DE genes with lower expression in LM of low tADG pigs was enriched in genes with functions related to muscle contraction, glucose metabolism, cytoskeleton organization, muscle development, and response to hormone stimulus (enrichment score > 1.3). The list of DE genes with higher expression in low tADG LM was enriched in genes with functions related to protein catabolism (enrichment score > 1.3). Analysis of known gene-gene interactions identified possible regulators of these differences in gene expression in LM of high and low tADG pigs; these include forkhead box O1 (FOXO1), growth hormone (GH1), and the glucocorticoid receptor (NR3C1). Differences in gene expression between poor transitioning pigs and their contemporaries indicate a shift to decreased protein synthesis, increased protein degradation, and reduced glucose metabolism in the LM of low tADG pigs.

The MC4R c.893G>A mutation: a marker for growth and leanness associated with boar taint odour in Belgian pig breeds.

Since surgical castration of male piglets without anaesthesia is under heavy societal pressure, finding a sustainable solution to reduce boar taint has become urgent. One way to circumvent this animal welfare violation is raising entire male pigs whilst selecting against the tainted phenotype through marker-assisted selection. Since slaughtering at a lower weight is often suggested to reduce boar taint, selection using a marker for that trait could be a promising strategy. Therefore, in this study a melanocortin 4 receptor (MC4R) mutation, frequently described in different pig breeds as marker for fat content, weight gain and feed intake, was examined in relation to boar taint in pig breeds used in Belgian pig farms. Although results suggest an association between this mutation and a boar taint odour score assigned by experts, no association was found between the mutation and the concentration of the individual chemical boar taint components androstenone, skatole and indole.

E. coli heat labile toxin (LT) inactivation by specific polyphenols is aggregation dependent.

Recently, polyphenol extracts were suggested to inhibit binding of Escherichia coli heat labile enterotoxin (LT) to its intestinal receptor GM1. Therefore, polyphenols are promising feed or food supplements to combat enterotoxigenic infections. Little is known of the precise mechanism, or the type of polyphenol required. Here, seven different polyphenols were tested in vitro (1) for inhibition of LT binding to GM1 (GM1-ELISA), (2) for LT inhibitory activity in the cAMP Vero-cell assay, and (3) by testing the aggregating properties of polyphenols with LT using molecular weight exclusion membrane filters, and by centrifugation techniques. Results showed only three out of seven polyphenols, pentagalloylglucose (PGG), epigallocatechingallate (EGCG) and gallocatechingallate (GCG), to effectively inhibit binding of LT to GM1, and to inhibit induction of cAMP in Vero cells, and that PGG is the most effective. Blocking of the GM1 receptor is unlikely as a mechanism because pre-incubation of GM1 with polyphenols had no effect. Co-incubation of polyphenols with forskolin did not interfere with cAMP production in Vero cells, showing that polyphenol activity is not directly related to cAMP. It is concluded that the inhibitory activities of these three polyphenols may coincide with the formation of large (>100 kDa) LT-polyphenol aggregates. Enterotoxin inactivation appears to require a minimum of two galloyl moieties in polyphenol structure and the pentagalloyl PGG is the most effective.

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: a gene expression study.

Diarrhoea in neonatal and early-weaned piglets due to enterotoxigenic Escherichia coli-F4 (ETEC-F4) is an important problem in the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13 has been found to be in close linkage to the susceptibility of piglets for ETEC-F4ab,ac. Potential candidate genes on SSC13 have been examined and although some polymorphisms were found to be in linkage disequilibrium with the phenotype, the causative mutation has not yet been found. In this study we are looking at the expression of porcine genes in relation to ETEC-F4ab,ac. With the aid of the Affymetrix GeneChip Porcine Genome Array we were able to find differentially expressed genes between ETEC-F4ab,ac receptor positive (Fab,acR(+)) piglets without diarrhoea and F4ab,acR(+) piglets with diarrhoea or F4ab,acR(-) animals. Since the susceptibility to ETEC-F4ab,ac was described as a Mendelian trait, it is not so surprisingly that only two differentially expressed genes, transferrin receptor (TFRC) and trefoil factor 1 (TFF1), came out of the analysis. Although both genes could pass for functional candidate genes only TFRC also mapped to the region on SSC13 associated with susceptibility for ETEC-F4, which makes TFRC a positional functional candidate gene. Validation by qRT-PCR confirmed the differential expression of TFRC and TFF1. In piglets without diarrhoea, the expression of both genes was higher in F4ab,acR(+) than in F4ab,acR(-) piglets. Similarly, TFRC and TFF1 expression in F4ab,acR(+) piglets without diarrhoea was also higher than in F4ab,acR(+) piglets with diarrhoea. Consequently, although both genes might not play a role as receptor for F4 fimbriae, they could be of great importance during an ETEC-F4 outbreak. An upregulation of TFRC can be a consequence of the piglets ability to raise an effective immune response. An elevation of TFF1, a protein involved in mucin formation, may also affect the piglet's capability to cope with ETEC bacteria, rather than being a receptor for its fimbriae.

Susceptibility of piglets to enterotoxigenic Escherichia coli is not related to the expression of MUC13 and MUC20.

Enterotoxigenic Escherichia coli (ETEC) is one of the most frequently isolated enteropathogens in production animals, especially pigs and calves. Economically, the swine industry is by far the most affected by infections with ETEC because of mortality, morbidity and decreased growth rate of newborn and early-weaned piglets. After ingestion by the animal, these bacteria attach themselves to specific receptors on the small intestinal epithelium by means of proteinaceous surface appendages, the fimbriae. The F4 fimbriae, which attach to the F4 receptor, are the most studied. The aim of our study was to investigate gene expression in the small intestine of piglets of MUC13 and MUC20 in relation to animals with a different treatment towards or a different reaction on ETEC-F4ac by means of quantitative reverse transcription chain reaction (qRT/PCR). MUC13 and MUC20 are positional candidate genes for this F4ac receptor and are located in the region on SSC13q41 that segregates with the susceptibility to ETEC-F4ac. The condition of the small intestine is crucial when examining expression differences between different samples. Therefore, the expression of two genes, fatty-acid binding protein 2, intestinal (FABP2) and pancreatitis-associated protein (PAP), now known as regenerating islet-derived 3 alpha (REG3A) in the small intestine was simultaneously checked. FABP2, a standard for epithelial content, reflects the state of damage, whereas REG3A is a measure for inflammation in the small intestine. The four different substudies presented here suggest that expression of MUC13 and MUC20 is not related to the susceptibility of piglets to ETEC-F4ac.

Indirect effect of IGF2 intron3 g.3072G>A mutation on prolificacy in sows.

A QTL located in the paternally expressed insulin-like growth factor 2 (IGF2) gene is known to increase muscle growth and reduce fat deposition in pigs. This makes the QTL in IGF2 a good marker for use in pig breeding programmes. However, care has to be taken as it is postulated that increased leanness and lowered fat deposition may have a negative effect on the prolificacy and longevity of sows. Selection of sire and dam lines for different alleles of the mutation in the paternally imprinted IGF2 gene could actually provide a solution to this problem. Therefore, in this study, the effect of the IGF2 QTL on prolificacy-related traits in sows was investigated. It was found that the paternal IGF2 wild-type allele was associated with higher reproduction performance in the sow. Moreover, it was also examined whether the difference in prolificacy in sows could be a consequence of differential IGF2 expression in the ovarian follicles of the sow or whether it is mainly a secondary effect caused by differences in fatness traits. Therefore, IGF2 expression was measured in follicles of different sizes from sows with different genotypes for the paternal IGF2 allele. It was observed that, however, while the size of the follicles was associated with follicular IGF2 expression level, the IGF2 genotype was not. It could be concluded that the difference in prolificacy of sows with a different paternal IGF2 genotype could be a secondary effect, resulting from differences in fat deposition.