Transcriptional alteration of p53 related processes as a key factor for skeletal muscle characteristics in <Emphasis Type="Italic">Sus scrofa</Emphasis>

The pig could be a useful model to characterize molecular aspects determining several delicate phenotypes because they have been bred for those characteristics. The Korean native pig (KNP) is a regional breed in Korea that was characterized by relatively high intramuscular fat content and reddish meat color compared to other western breeds such as Yorkshire (YS). YS grew faster and contained more lean muscle than KNP. We compared the KNP to Yorksire to find molecular clues determining muscle characteristics. The comparison of skeletal gene expression profiles between these two breeds showed molecular differences in muscle. We found 82 differentially expressed genes (DEGs) defined by fold change (more than 1.5 fold difference) and statistical significance (within 5% of false discovery rate). Functional analyses of these DEGs indicated up-regulation of most genes involved in cell cycle arrest, down-regulation of most genes involved in cellular differentiation and its inhibition, down-regulation of most genes encoding component of muscular-structural system, and up-regulation of most genes involved in diverse metabolism in KNP. Especially, DEGs in above-mentioned categories included a large number of genes encoding proteins directly or indirectly involved in p53 pathway. Our results indicated a possible role of p53 to determine muscle characteristics between these two breeds.     

Transcriptomic investigation of meat tenderness in two Italian cattle breeds

Our objectives for this study were to understand the biological basis of meat tenderness and to provide an overview of the gene expression profiles related to meat quality as a tool for selection. Through deep mRNA sequencing, we analyzed gene expression in muscle tissues of two Italian cattle breeds: Maremmana and Chianina. We uncovered several differentially expressed genes that encode for proteins belonging to a family of tripartite motif proteins, which are involved in growth, cell differentiation and apoptosis, such as TRIM45, or play an essential role in regulating skeletal muscle differentiation and the regeneration of adult skeletal muscle, such as TRIM32. Other differentially expressed genes (SCN2B, SLC9A7 and KCNK3) emphasize the involvement of potassium–sodium pumps in tender meat. By mapping splice junctions in RNA‐Seq reads, we found significant differences in gene isoform expression levels. The PRKAG3 gene, which is involved in the regulation of energy metabolism, showed four isoforms that were differentially expressed. This distinct pattern of PRKAG3 gene expression could indicate impaired glycogen storage in skeletal muscle, and consequently, this gene very likely has a role in the tenderization process. Furthermore, with this deep RNA‐sequencing, we captured a high number of expressed SNPs, for example, we found 1462 homozygous SNPs showing the alternative allele with a 100% frequency when comparing tender and tough meat. SNPs were then classified into categories by their position and also by their effect on gene coding (174 non‐synonymous polymorphisms) based on the available UMD_3.1 annotations.     

Genetic diversity between Piedmontese, Maremmana, and Podolica cattle breeds

One hundred twenty animals of three native Italian cattle breeds, Piedmontese, Maremmana, and Podolica, were genetically characterized at 21 microsatellite loci located on 13 chromosomes. Allele numbers ranged from 3 to 19; average gene diversity ranged from 0.206 to 0.878 (average 0.738). The breed that preserved the highest genetic variability was the Podolica, where the chosen markers show the highest gene diversity (0.741) and the highest heterozygosity (0.155). The lowest inbreeding rate (0.102) was registered by the Piedmontese. Genetic distances were 0.069 (Piedmontese versus Maremmana), 0.050 (Piedmontese versus Podolica), and 0.041 (Maremmana versus Podolica) and reflect the different phylogenetic origins of the breeds: Maremmana and Podolica originated from the Grey Steppe cattle group, while Piedmontese belongs to the Northern Italy Lowland cattle group. Observed heterozygosity was not significantly different from expected in any of the breeds, which is an indication that they maintain a random mating structure. The probabilistic assignment of all sampled individuals to three theoretical populations, on the basis of allele frequencies, indicated that 82% of Piedmontese, 66% of Maremmana, and 33% of Podolica can be assigned to the appropriate breed with a probability higher than 90%. This result very well reflects the impact of the selection activity on the breed genetic structure. The chosen microsatellites proved to be a good tool for describing the correct reality of the analyzed populations, but they are not sufficient to discriminate between breeds.     

Genetic Variability and Linkage Disequilibrium Patterns in the Bovine <Emphasis Type="Italic">DNAJA1</Emphasis> Gene

Correlation between expression level of the bovine DNAJA1 gene and meat tenderness was recently found in Charolais longissimus thoracis muscle samples, suggesting that this gene could play an important role in meat tenderness. Here, we report the validation of polymorphisms within the bovine DNAJA1 gene, and the haplotype variability and extent of linkage disequilibrium in the three main French beef breeds (Blonde d’Aquitaine, Charolais, Limousin). Genotyping 18 putative SNPs revealed that 16 SNPs were polymorphic within the breeds tested. Two SNPs were removed from further analyses as one SNP had a low genotyping call rate, while the other SNP was not in Hardy–Weinberg equilibrium. The degree of heterozygosity observed for the remaining 14 SNPs varied between breeds, with Charolais being the breed with the highest genetic variation and Blonde d’Aquitaine the lowest. Linkage disequilibrium and haplotype structure of DNAJA1 were different between breeds. Eighteen different haplotypes, including three shared by all breeds, were discovered, and two to three tag SNPs (depending on the breed) are sufficient to capture all the genetic variability seen in these haplotypes. The results of this study will facilitate the design of optimal future association studies evaluating the role of the DNAJA1 gene in meat tenderness.     

Early antibody response against <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subspecies <Emphasis Type="Italic">paratuberculosis</Emphasis> antigens in subclinical cattle

Background  

Our laboratories have previously reported on the experimental infection of cattle with Mycobacterium avium subsp paratuberculosis (M. paratuberculosis) using an intratonsillar infection model. In addition, we have recently developed a partial protein array representing 92 M. paratuberculosis coding sequences. These combined tools have enabled a unique look at the temporal analysis of M. paratuberculosis antigens within the native host. The primary objective of this study was to identify M. paratuberculosis antigens detected by cattle early during infection. A secondary objective was to evaluate the humoral immune response in cattle during the initial year of infection.     

New SNP of the porcine Perilipin 2 (<Emphasis Type="Italic">PLIN2</Emphasis>) gene,association with carcass traits and expression analysis in skeletal muscle

PLIN2 (perilipin 2) is a cytosolic protein that promotes the formation and stabilization of the intracellular lipid droplets, organelles involved in the storage of lipid depots. Porcine PLIN2 gene represents a biological and positional candidate for fat deposition, a polygenic trait that affects carcass and meat quality. The aim of the present study was to screen PLIN2 gene for polymorphisms, to evaluate the association with carcass quality traits, and to investigate the gene expression in skeletal muscle. Six new single nucleotide polymorphisms (SNP) were detected by sequencing 32 samples from five pig breeds (Italian Large White, Italian Duroc, Italian Landrace, Belgian Landrace, Pietrain). Two SNP localized in introns, two in the 3′-untranslated region (UTR), and two missense SNP were found in exons. A 3′-UTR mutation (GU461317:g.98G>A), genotyped in 290 Italian Duroc pigs by High Resolution Melting, resulted significantly associated (P < 0.01) with average daily gain, feed conversion ratio, lean cuts and hams weight estimated breeding values. PLIN2 gene expression analysis in skeletal muscle of Italian Large White and Italian Duroc pigs divergent for backfat thickness and visible intermuscular fat showed a trend of higher expression level in pigs with higher intermuscular fat. These results suggest that PLIN2 can be a marker for carcass quality in pigs. Further investigation at both gene and protein level could elucidate its role on fat deposition.     

The porcine <Emphasis Type="Italic">TBC1D1</Emphasis> gene: mapping,SNP identification,and association study with meat,carcass and production traits in Italian heavy pigs

TBC1D1 [TBC1 (tre-2/USP6, BUB2, cdc16) domain family, member 1] is a Rab-GTPase-activating related protein implicated in regulating the trafficking of glucose transporter 4 (GLUT4 or SLC2A4) storage vesicles to the cell surface in response to insulin and AMPK-activating stimuli in skeletal muscle. Mutations in the human and mouse TBC1D1 genes confer risk of obesity or leanness. We identified five single nucleotide polymorphisms (SNPs) in the porcine TBC1D1 gene. One of them (FN677935:g.219G>A) was genotyped either by high resolution melting and PCR-RFLP analyses to study allele frequencies in a few pig breeds and evaluate association with meat production and carcass traits in five groups of sib-tested pigs of Italian Large White and Italian Duroc breeds. The g.219G>A SNP was associated (P < 0.05) with ham weight, back fat thickness and lean cuts content in Italian Large White and with visible intermuscular fat in Italian Duroc pigs.     

Chianina beef tenderness investigated through integrated Omics

In the present study we performed an integrated proteomics, interactomics and metabolomics analysis of Longissimus dorsi tender and tough meat samples from Chianina beef cattle. Results were statistically handled as to obtain Pearson's correlation coefficients of the results from Omics investigation in relation to canonical tenderness-related parameters, including Warner Bratzler shear force, myofibrillar degradation (at 48 h and 10 days after slaughter), sarcomere length and total collagen content. As a result, we could observe that the tender meat group was characterized by higher levels of glycolytic enzymes, which were over-phosphorylated and produced accumulation of glycolytic intermediates. Oxidative stress promoted meat tenderness and elicited heat shock protein responses, which in turn triggered apoptosis-like cascades along with PARP fragmentation. Phosphorylation was found to be a key process in post mortem muscle conversion to meat, as it was shown not only to modulate glycolytic enzyme activities, but also mediate the stability of structural proteins at the Z-disk. On the other hand, phosphorylation of HSPs has been supposed to alter their functions through changing their affinity for target interactors. Analogies and breed-specific differences are highlighted throughout the text via a direct comparison of the present results against the ones obtained in a parallel study on Maremmana Longissimus dorsi. It emerges that, while the main cornerstones and the final outcome are maintained, post mortem metabolism in tender and tough meat yielding individuals is subtly modulated via specific higher levels of enzymes and amino acidic residue phosphorylation in a breed-specific fashion, and whether calcium homeostasis dysregulation was a key factor in Maremmana, higher early post mortem phosphocreatine levels in the Chianina tender group could favor a slower and prolonged glycolytic rate, prolonging the extent of the minimum hanging period necessary to obtain tender meat from this breed by a few days.     

Deletion of methylglyoxal synthase gene (<Emphasis Type="Italic">mgsA</Emphasis>) increased sugar co-metabolism in ethanol-producing <Emphasis Type="Italic">Escherichia coli</Emphasis>

The use of lignocellulose as a source of sugars for bioproducts requires the development of biocatalysts that maximize product yields by fermenting mixtures of hexose and pentose sugars to completion. In this study, we implicate mgsA encoding methylglyoxal synthase (and methylglyoxal) in the modulation of sugar metabolism. Deletion of this gene (strain LY168) resulted in the co-metabolism of glucose and xylose, and accelerated the metabolism of a 5-sugar mixture (mannose, glucose, arabinose, xylose and galactose) to ethanol.     

Characterization of the porcine differentially expressed <Emphasis Type="Italic">PDK4</Emphasis> gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.     

Characterization of the expression profile of <Emphasis Type="Italic">calpain</Emphasis>-<Emphasis Type="Italic">3</Emphasis> (<Emphasis Type="Italic">CAPN3</Emphasis>) gene in chicken

Calpain-3 is a skeletal muscle-specific protease and participates in the regulation of myogenesis. In this study, we quantified the expression of calpain-3 (CAPN3) mRNA in a Chinese local chicken breed (Sichuan Mountainous Black-boned chicken [MB]), to discern the tissue and ontogenic expression pattern. Meanwhile, we compared the CAPN3 mRNA expression pattern in MB chicken at 10 weeks with a commercial meat type chicken line (S01) of the same age to identify the unique expression pattern under different genetic background. A real time quantitative PCR (qRT-PCR) assay was developed for an accurate measurement of its expression in various tissues from chickens at different ages (0, 2, 4, 6, 8, 10, and 12 weeks). Expression of the CAPN3 mRNA was detected in the selected tissues, regardless of age. The breast muscle and leg muscle tissues had a significantly higher expression than the other tissues from the same individual (P < 0.01). Overall, the CAPN3 mRNA level exhibited a “rise-decline” developmental change in detected tissues except for brain. The S01 chicken had a higher expression of the CAPN3 mRNA in detected tissues than the MB chicken at 10 weeks. The present expression data of chicken CAPN3 gene may provide some information to shed light on the tissue and ontogenic expression pattern during chicken development.     

Sexual Dimorphism in the Pelvis of <Emphasis Type="Italic">Microcebus</Emphasis>

Pelvic sexual dimorphism occurs in many anthropoid species and is often attributed to obstetric selection on female pelvic morphology. Few studies of pelvic dimorphism have included strepsirrhine taxa, which typically have relatively smaller infants than those of anthropoids. Because smaller female primates give birth to relatively larger infants, it is possible that the pelves of Microcebus, the smallest extant primate genus, will show some evidence of selection on obstetric adequacy. A comparison of adult female and neonatal body masses indicates that individual neonatal Microcebus are relatively large compared to adult female body mass, even though members of the taxon frequently produce twins. I examined variation in the bony pelvis within a sample of Microcebus. I measured specimens from a single locality, which probably represent 1 population. I measured 8 pelvic and 3 femoral variables to investigate skeletal size and pelvic size and shape dimorphism. Females significantly exceed males in absolute values of sacral width, pelvic height, pubic length, and distances from the pubic symphysis to the ischial tuberosity and points on the sacrum. Measurements of the femur are not significantly greater in females, suggesting that the pelvic differences are not due to skeletal size dimorphism. Significant pelvic shape or ratio differences, calculated via the geometric mean of 5 variables as the denominator, included greater relative pubic length and sacral width in females. Hence selection for obstetric adequacy may occur in the extremely small-bodied Microcebus.     

Detection of <Emphasis Type="Italic">Giardia,Entamoeba,</Emphasis> and <Emphasis Type="Italic">Cryptosporidium</Emphasis> in unprocessed food items from northern India

This study was conducted to evaluate the load of three food- and water-borne parasites, namely, Giardia, Entamoeba, and Cryptosporidium on food items that are consumed either raw or in an unprocessed state in the northern parts of India. A two-step diagnostic method was employed to assess the presence of Giardia and Cryptosporidium, the immunofluorescence assay (IFA) combined with the polymerase chain reactiion (PCR assay), whereas a Tech lab diagnostic kit in combination with PCR assay was used for accurate detection of Entamoeba histolytica in the samples. The methods for isolation and enrichment of cysts/oocysts from the various food items were tested and discussed here. Our results showed that the overall spectrum of incidence of the three parasites on food items in decreasing order were Giardia > Entamoeba > Cryptosporidium. When data were subjected to the chi-square test, the prevalence of all three parasites was found to be independent of the food items. To determine whether the presence of two types of parasites in a food item is uniform, a Poisson distribution test was conducted. On comparing the intensity of occurrence of the different parasites in various food items, it was observed that the occurrence of Giardia and Entamoeba was not of the same order at 5% level of significance only in case of samples of raw meat and milk. This confirmed that a high number of raw or unpasteurized milk and meat samples are more likely to be contaminated with Giardia than with Entamoeba. Therefore, our observations point to the unhygienic practices of food handlers being a major contributor in the transmission of parasites to unprocessed food products.     

Polymorphism of the <Emphasis Type="Italic">BoLA-DRB3</Emphasis> gene in the Mongolian,Kalmyk, and Yakut cattle breeds

Polymorphism of the BoLA-DRB3 gene was studied with the use of the PCR-RFLP technique in three cattle breeds (Mongolian, Kalmyk, and Yakut) representing the Bos taurus turano-mongolicus group. 35 BoLA-DRB3.2 alleles were detected in the Mongolian breed and 34 alleles in the Kalmyk breed. The frequencies of alleles in both populations are distributed rather evenly: the frequencies of the most widely represented alleles (*18, *20, and *28) in the Mongolian cattle varied from 7.75 to 8.45%. The most frequent alleles in the Kalmyk cattle were *28 (14.52%), *24 (7.26%), and *12 (6.45%). Only five alleles were identified in the Yakut cattle breed. The prevailing allele was *29 (77.3%); a relatively frequent allele was *1 (13.1%), and the remaining three alleles constituted only 9.6%. Such a low level of diversity of BoLA-DRB3 gene alleles was not observed earlier in any other cattle breed. The Mongolian and Kalmyk breeds showed a wide diversity of BoLA-DRB3 genotypes (56 and 51 genotypes, respectively) and a high level of expected heterozygosity (H _e = 0.953 and 0.946, respectively). Both breeds had a deficiency of heterozygotes (Mongolian cattle: H _o = 0.775, D = −0.187; Kalmyk cattle: H _o = 0.708, D = −0.252). A low level of genotypic diversity for the BoLA-DRB3 locus (only seven genotypes; the frequency for the genotype *29/*29 is 71.4%) and a very low level of observed heterozygosity (H _o = 0.12) were revealed in the Yakut breed. BoLA-DRB3.2 alleles associated with resistance to persistent lymphocytosis caused by the bovine leukemia virus (total frequencies 15.49 and 24.19%) and to various forms of mastitis (total frequencies 12.68 and 20.96%, respectively) were identified in the Mongolian and Kalmyk animals. In the Yakut breed, alleles associated with resistance to diseases are represented only by the BoLA-DRB3.2 allele *7 (1.2%). Thus, the Mongolian and Kalmyk cattle breeds are characterized by a wide diversity of alleles and genotypes for the BoLA-DRB3 gene. In contrast, the population of Yakut cattle from the Verkhoyanskii region of the Republic of Sakha has a poor diversity of alleles and genotypes for the BoLA-DRB3 gene and a very low level of heterozygosity, suggesting an unfavorable state of the population that is probably caused by inbreeding depression due to a long-term isolation and a small number of animals.