Characterization of Global DNA Methylation in Different Gene Regions Reveals Candidate Biomarkers in Pigs with High and Low Levels of Boar Taint

DNA methylation of different gene components, including different exons and introns, or different lengths of exons and introns is associated with differences in gene expression. To investigate the methylation of porcine gene components associated with the boar taint (BT) trait, this study used reduced representation bisulfite sequencing (RRBS) data from nine porcine testis samples in three BT groups (low, medium and high BT). The results showed that the methylation levels of the first exons and first introns were lower than those of the other exons and introns. The first exons/introns of CpG island regions had even lower levels of methylation. A total of 123 differentially methylated promoters (DMPs), 194 differentially methylated exons (DMEs) and 402 differentially methylated introns (DMIs) were identified, of which 80 DMPs (DMP-CpGis), 112 DMEs (DME-CpGis) and 166 DMIs (DMI-CpGis) were discovered in CpG islands. Importantly, GPX1 contained one each of DMP, DME, DMI, DMP-CpGi, DME-CpGi and DMI-CpGi. Gene-GO term relationships and pathways analysis showed DMP-CpGi-related genes are mainly involved in methylation-related biological functions. In addition, gene–gene interaction networks consisted of nodes that were hypo-methylated GPX1, hypo-methylated APP, hypo-methylated ATOX1, hyper-methylated ADRB2, hyper-methylated RPS6KA1 and hyper-methylated PNMT. They could be used as candidate biomarkers for reducing boar taint in pigs, after further validation in large cohorts.

Does the Pre-Ovulatory Pig Oviduct Rule Sperm Capacitation In Vivo Mediating Transcriptomics of Catsper Channels?

Spermatozoa need to conduct a series of biochemical changes termed capacitation in order to fertilize. In vivo, capacitation is sequentially achieved during sperm transport and interaction with the female genital tract, by mechanisms yet undisclosed in detail. However, when boar spermatozoa are stored in the tubal reservoir pre-ovulation, most appear to be in a non-capacitated state. This study aimed at deciphering the transcriptomics of capacitation-related genes in the pig pre-ovulatory oviduct, following the entry of semen or of sperm-free seminal plasma (SP). Ex-vivo samples of the utero-tubal junction (UTJ) and isthmus were examined with a microarray chip (GeneChip® Porcine Gene 1.0 ST Array, Thermo Fisher Scientific) followed by bioinformatics for enriched analysis of functional categories (GO terms) and restrictive statistics. The results confirmed that entry of semen or of relative amounts of sperm-free SP modifies gene expression of these segments, pre-ovulation. It further shows that enriched genes are differentially associated with pathways relating to sperm motility, acrosome reaction, single fertilization, and the regulation of signal transduction GO terms. In particular, the pre-ovulation oviduct stimulates the Catsper channels for sperm Ca2+ influx, with AKAPs, CATSPERs, and CABYR genes being positive regulators while PKIs and CRISP1 genes appear to be inhibitors of the process. We postulate that the stimulation of PKIs and CRISP1 genes in the pre-ovulation sperm reservoir/adjacent isthmus, mediated by SP, act to prevent premature massive capacitation prior to ovulation.

The Transcriptome of Pig Spermatozoa, and Its Role in Fertility

In the study presented here we identified transcriptomic markers for fertility in the cargo of pig ejaculated spermatozoa using porcine-specific micro-arrays (GeneChip® miRNA 4.0 and GeneChip® Porcine Gene 1.0 ST). We report (i) the relative abundance of the ssc-miR-1285, miR-16, miR-4332, miR-92a, miR-671-5p, miR-4334-5p, miR-425-5p, miR-191, miR-92b-5p and miR-15b miRNAs, and (ii) the presence of 347 up-regulated and 174 down-regulated RNA transcripts in high-fertility breeding boars, based on differences of farrowing rate (FS) and litter size (LS), relative to low-fertility boars in the (Artificial Insemination) AI program. An overrepresentation analysis of the protein class (PANTHER) identified significant fold-increases for C-C chemokine binding (GO:0019957): CCR7, which activates B- and T-lymphocytes, 8-fold increase), XCR1 and CXCR4 (with ubiquitin as a natural ligand, 1.24-fold increase), cytokine receptor activity (GO:0005126): IL23R receptor of the IL23 protein, associated to JAK2 and STAT3, 3.4-fold increase), the TGF-receptor (PC00035) genes ACVR1C and ACVR2B (12-fold increase). Moreover, two micro-RNAs (miR-221 and mir-621) were down- and up-regulated, respectively, in high-fertility males. In conclusion, boars with different fertility performance possess a wide variety of differentially expressed RNA present in spermatozoa that would be attractive targets as non-invasive molecular markers for predicting fertility.

Detection of Porcine DNA in Processed Beef Products Using Real Time – Polymerase Chain Reaction

Meat is one of food materials which has protein source and mostlyconsumed by non-vegetarian. Consuming halal food is an obligation for every Muslim. Meat processed products usually contaminated by pork. One of technique that is often chosen as an authentication process for proofing halalness of the product is PCR technique, one of PCR technique which most commonly used is RT-PCR. RT-PCR technique was chosen as identification method because it has high accuration for detection of porcine DNA in fresh meat and processed products. RT-PCR is the amplification technique in the specific regions that are restricted by two oligonucleotide with the help of polymerase enzymes. Annealing is the first process of RT-PCR analysis who was primary attachment to the DNA template that determines the specificity and amount of DNA produced. In this study, extraction kit and detection kit were used for analysis Porcine DNA in meatballs. The results obtained from this study were from whole DNA samples, which had DNA purity ranging from 1.82 to 1.93. From the all samples three of them containing porcine DNA. The positive samples shown from amplification curves who was specifically formed when probes reacts with porcine gene.

Amino acids metabolism and degradation is regulated during porcine oviductal epithelial cells (OECs) primary culture in vitro – a signaling pathways activation approach

The ovary is part of the reproductive system, possessing very important functions in the reproduction process (ovum and embryo transfer, providing a suitable environment for sperm capacitation, etc.). There are two types of cells in the fallopian tubes: alveolar and secretive cells. These study shows the metabolic processes in pig oviductal epithelial cells associated with the activation of signaling pathways of amino acids metabolism and degradation during long-term in vitro culture. Oviductal epithelial cells from 45 colonies in the anestrous phase of the estrous cycle have been utilized in this study. RNA extract from the OEC primary cultures was pooled after 24h, 7days, 15 days and 30 days from the beginning of culture and the transcriptome investigated by Affymetrix® Porcine Gene 1.1 ST. From the whole transcript that consisted of 2009 different genes, 1537 were upregulated and 995 were downregulated after 7 days of culture, 1471 were upregulated and 1061 were downregulated after 15 days of culture and 1329 were upregulated and 1203 were downregulated after 30 days of culture. The results of these studies provide, for the first time, information on the activation of metabolic pathways of amino acids such as valine, leucine, isoleucine, cysteine, and methionine in the investigated tissue. They also indicate genes that may be OECs-specific genetic markers that are expressed or upregulated during long-term in vitro culture.

A Systems Biology Approach Using Transcriptomic Data Reveals Genes and Pathways in Porcine Skeletal Muscle Affected by Dietary Lysine

Nine crossbred finishing barrows randomly assigned to 3 dietary treatments were used to investigate the effects of dietary lysine on muscle growth related metabolic and signaling pathways. Muscle samples were collected from the longissimus dorsi of individual pigs after feeding the lysine-deficient, lysine-adequate, or lysine-excess diet for 5 weeks, and the total RNA was extracted afterwards. Affymetrix Porcine Gene 1.0 ST Array was used to quantify the expression levels of 19,211 genes. A total of 674 transcripts were differentially expressed (P ≤ 0.05); 60 out of 131 transcripts (P ≤ 0.01) were annotated in the NetAffx database. Ingenuity pathway analysis showed that dietary lysine deficiency may lead to (1) increased muscle protein degradation via the ubiquitination pathway as indicated by the up-regulated DNAJA1, HSP90AB1 and UBE2B mRNA, (2) reduced muscle protein synthesis via the up-regulated RND3 and ZIC1 mRNA, (3) increased serine and glycine synthesis via the up-regulated PHGDH and PSPH mRNA, and (4) increased lipid accumulation via the up-regulated ME1, SCD, and CIDEC mRNA. Dietary lysine excess may lead to (1) decreased muscle protein degradation via the down-regulated DNAJA1, HSP90AA1, HSPH1, and UBE2D3 mRNA, and (2) reduced lipid biosynthesis via the down-regulated CFD and ME1 mRNA.

Molecular cloning, sequence identification, polymorphism and association of the porcine <i>SPATS2L</i> gene

Spermatogenesis-associated, serine-rich 2-like (SPATS2L) is an important reproduction-related gene. In this study, the full-length cDNA sequence of the porcine SPATS2L gene was cloned through the RACE (rapid amplification of cDNA ends) method. The porcine SPATS2L gene encodes a protein of 559 amino acids which shares high homology with the SPATS2L proteins of seven species: dog (94 %), white-tufted-ear marmoset (91 %), human (92 %), rhesus monkey (92 %), horse (92 %), rat (88 %) and mouse (88 %). This novel porcine gene was assigned to GeneID: 100415809. The phylogenetic analysis revealed that the porcine SPATS2L gene has a close genetic relationship with the canine SPATS2L gene. PCR-Pst I-RFLP was established to detect GU474997:c.1687 C > T substitution of porcine SPATS2L gene mRNA, and eight pig breeds displayed obvious genotype and allele frequency differences at this mutation locus. Association of this single-nucleotide polymorphism (SNP) with litter size traits was assessed in Large White (n = 100) and Landrace (n = 100) pig populations, and results demonstrated that this polymorphic locus was significantly associated with the litter size of all parities in Large White sows and Landrace sows (P < 0.01). Therefore, SPATS2L gene could be an useful candidate gene in selection for increasing litter size in pigs.