Review: Effect of essential fatty acids and conjugated linoleic acid on the adaptive physiology of dairy cows during the transition period

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

MicroRNA profiling of subcutaneous adipose tissue in periparturient dairy cows at high or moderate body condition

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

Overproduction and Characterization of the Bacillus subtilis Anti-sigma Factor FlgM

FlgM is an anti-sigma factor of the flagellar-specific sigma (sigma) subunit of RNA polymerase in Bacillus subtilis, and it is responsible of the coupling of late flagellar gene expression to the completion of the hook-basal body structure. We have overproduced the protein in soluble form and characterized it. FlgM forms dimers as shown by gel exclusion chromatography and native polyacrylamide gel electrophoresis and interacts in vitro with the cognate sigmaD factor. The FlgM.sigmaD complex is a stable heterodimer as demonstrated by gel exclusion chromatography, chemical cross-linking, native polyacrylamide gel electrophoresis, and isoelectric focusing. sigmaD belongs to the group of sigma factors able to bind to the promoter sequence even in the absence of core RNA polymerase. The FlgM.sigmaD complex gave a shift in a DNA mobility shift assay with a probe containing a sigmaD-dependent promoter sequence. Limited proteolysis studies indicate the presence of two structural motifs, corresponding to the N- and C-terminal regions, respectively

Expression Analysis of MicroRNAs in FFPE samples of canine cutaneous and oral melanoma by RT-qPCR

MicroRNA (miRNA), a class of small, non-coding RNA - regulating post-transcriptionally protein expression - are emerging as clinical biomarkers in many pathologies, including cancer [1]. Since miRNA are supposed to represent fundamental key regulators, better understanding of melanoma biology is essential to improve staging and therapy. The aim of the study was to investigate whether miRNA expression can vary in canine melanoma samples derived from formalin-fixed-paraffin-embedded (FFPE) tissues. Experimental design of the study included three groups, each one composed of 7 animals: i) control healthy skin group ii) oral melanoma group iii) skin melanoma group. The histhopatology and immunoistochemistry details of dogs included in the study are previously reported  [2]. Two tissue slides were used for miRNA extraction. The expression levels of seven miRNA - miR-145, miR-146a, miR-425-5p, miR-223, miR-365, miR-155 and miR-134 - were detected and assessed by qPCR using TaqMan® probes [3-5]. Five miRNA were significantly up-(n=3) or down-(n=2) regulated. In details, miR-146a and miR-155 abundance was increased as compared with control in both oral and skin melanoma (Fig 1 B,E) (p = 0.004 and 0.014 and p = 0.043 and 0.035 respectively), while the levels of miR-145 and miR-365 were lower (Fig 1 A,D) (p = 0.018 and 0.008 and p = 0.01 and 0.028, respectively). MiR-425-5p was upregulated (p = 0.039) only in skin melanoma (Fig. 1 C). Furthermore, functional analysis, carried out using miRNet web-based tool, showed that 76 genes related to cancer-associated pathways were possible target of these five microRNA (p = 6.95E-9); in particular, 21 target genes were associated with melanoma (p = 1.47E-5), including BRAF and CDK, E2F, FGF and PIK3 families. In conclusion, miR-145, miR-146a, miR-425-5p, miR-365 and miR-155 are differentially expressed in melanoma and healthy FFPE samples, suggesting that they may play a role in canine melanoma pathogenesis and/or progression

Evaluation of quantity and purity of miRNAs extracted from different matrices collected from dogs with Mast Cell Tumours.

MicroRNAs (miRNAs) are a class of short non-coding RNA, which interact with the 3’ UTR region of complementary mRNA to decrease or inhibit the translation of proteins (Lai, 2002). MiRNAs regulate pathways in various pathophysiological status, and are regarded as biomarkers for early diagnosis of several diseases, including cancer (Di Leva et al., 2014).The study aims to evaluate the quality and purity of miRNAs extracted from a) 11 archival Formalin Fixed and Paraffin Embedded (FFPE) samples of Mast Cell Tumour (MCT) at stage I, II, III and IV, and 8 intra-patient healthy controls; b) samples collected during surgery, including 6 samples of saliva, primary tumour biopsy and serum/plasma. The quality of miRNA largely influence the downstream experiments, and must be carefully evaluated before performing for examples, the sequencing reaction. MiRNA extraction was carried out using commercial kits (Qiagen) and quantify using Small RNA Kit (Agilent) on Agilent 2100 Bioanalyzer. The results showed that the concentration of miRNAs from FFPE, saliva,  primary tumor biopsy and serum was acceptable with a Median (Me)= 56,91 ng/ml, Me=10,30 ng/ml, Me=3,44 ng/ml and  Me=0,71 ng/ml, and a miRNA/Small RNA ratio of 48%, 61%, 17% and 76%, respectively. The concentration of miRNAs from plasma was not detectable. Studies reveal that plasma ranks as the first choice source for diagnostic purpose, much more than serum (Aung et al., 2014), but the debate remains open and subsequent analyses are needed.The concentration of miRNAs from FFPE and saliva samples is higher than that from other matrices. Possible explanations include a) different quantity and quality of starting materials; b) nucleic acids fragmentation, due to the formalin fixation and paraffin embedded procedure; c) presence of nucleases in saliva, which produce small fragments recognized as miRNAs or smallRNAs.In conclusion, the quantity and the purity of miRNAs, obtained using Qiagen commercial kits, are reliable for further NGS analysis

MicroARNs como biomarcadores de salud y bienestar animal en cerdos

Los microARNs (miARNs) son pequeñas moléculas de ARN no codificante muy conservadas que intervienen en una amplia gama de procesos biológicos mediante la regulación postranscripcional de la expresión génica. Un aspecto intrigante en la identificación de estas moléculas como biomarcadores se deriva de su papel en la comunicación entre células, su secreción activa desde las células al medio extracelular, su alta estabilidad en los fluidos corporales y su facilidad de obtención.Todas estas características confieren a los miARNs el potencial para convertirse en una herramienta no invasiva que permita evaluar el bienestar animal en condiciones de estrés metabólico, ambiental y de manejo. Esta revisión ofrece una visión general de los conocimientos actuales sobre el uso potencial de miARNs tisulares y/o circulantes como biomarcadores para la evaluación del estado de salud y bienestar en el porcino

Widespread extrahepatic expression of acute-phase proteins in chicken (Gallus gallus) tissues

Acute Phase Proteins (APP) are plasma proteins that can modify their expression in response toinflammation caused by tissue injury, infections, immunological disorders, or stress. Although APP areproduced mainly in liver, extrahepatic production has been described (Marques et al., 2016; Lecchi etal., 2012). The aim of this work was to study the extrahepatic gene expression of five APP, namely α1-acid glycoprotein (AGP), Serum amyloid A (SAA), Haptoglobin-like protein (PIT54), C-rective protein(CRP) and Ovotransferrin (OVT) (O'Reilly and Eckersall, 2014) in different healthy chicken (Gallus gallus)tissues by quantitative real time PCR (qPCR) and immunohistochemistry to detect the precise locationof the proteins.APP gene expression was higher in liver compared with other tissues. mRNA coding for CRP, OVT andSAA was detected in all tissues involved in this study with a higher expression in gastrointestinal tract,respiratory system and lymphatic system. SAA expression was particularly high in cecal tonsil, lung,spleen and meckel’s diverticulum, whereas OVT showed a high expression in lung, bursa of Fabricius,pancreas, brain and adipose tissue. AGP and PIT54 was also detected in pericardial adipose tissue,spleen, kidney, lung, mucosa of proventriculus, mucosa of gizzard and pancreas but, oppositely to SAA,their mRNA was not detected in meckel’s diverticulum, cecal tonsil and bursa of Fabricius. These resultssuggest that each tissue is able to express different amount of APP even in healthy conditions andmount a local acute phase reaction. Immunohistochemistry to detect the precise location for AGP, OVTand SAA using available antibodies is ongoing

The prevalence and risk factors of subclinical mastitis in water buffalo (Bubalis bubalis) in Bangladesh

Subclinical mastitis (SCM) in water buffalo is responsible for reduced milk yield and quality. This cross-sectional study was carried out to a) estimate the prevalence of SCM, b) identify risk factors associated with SCM, and c) identify farm-level risk factors associated with bulk milk somatic cell count (BMSCC). The buffalo farms included in this study represented five rearing systems: free-range, semi-free-range, household, semi-intensive, and intensive, providing a total of 3491 functional quarters of 880 lactating buffalo on 248 farms. The California mastitis test score was used to identify SCM. Bulk milk samples (n = 242) were used for farm-level BMSCC. Quarter and buffalo-level risk factors for SCM were measured using questionnaires and observations. The overall SCM prevalence was high at 27.9% at the quarter-level (25th and 75th percentiles: 8.3% and 41.7%) and 51.5% at buffalo-level (25th and 75th percentiles: 33.3% and 66.7%). The geometric mean BMSCC was 217,000 cells/mL of milk (ranging from 36,000-1,213,000 cells/mL), which is low on average, but some farms could improve substantially. The buffalo rearing system, udder location (left versus right), teat shape, udder asymmetry, number of milkers, and having a quarantine facility were associated with buffalo udder health. Our findings suggest that mainly using free-range rearing systems may help decrease the prevalence of SCM primarily by employing buffalo breeding and better farm biosecurity, and udder health control strategies can be designed based on our findings