The downregulation of genes encoding muscle proteins have a potential role in the development of scrotal hernia in pigs.

BACKGROUND: Testicular descent is a physiological process regulated by many factors. Eventually, disturbances in the embryological/fetal development path facilitate the occurrence of scrotal hernia, a congenital malformation characterized by the presence of intestinal portions within the scrotal sac due to the abnormal expansion of the inguinal ring. In pigs, some genes have been related to this anomaly, but the genetic mechanisms involved remain unclear. This study aimed to investigate the expression profile of a set of genes potentially involved with the manifestation of scrotal hernia in the inguinal ring tissue. METHODS AND RESULTS: Tissue samples from the inguinal ring/canal of normal and scrotal hernia-affected male pigs with approximately 30 days of age were used. Relative expression analysis was performed using qPCR to confirm the expression profile of 17 candidate genes previously identified in an RNA-Seq study. Among them, the Myosin heavy chain 1 (MYH1), Desmin (DES), and Troponin 1 (TNNI1) genes were differentially expressed between groups and had reduced levels of expression in the affected animals. These genes encode proteins involved in the formation of muscle tissue, which seems to be important for increasing the resistance of the inguinal ring to the abdominal pressure, which is essential to avoid the occurrence of scrotal hernia. CONCLUSIONS: The downregulation of muscular candidate genes in the inguinal tissue clarifies the genetic mechanisms involved with this anomaly in its primary site, providing useful information for developing strategies to control this malformation in pigs and other mammals.

Inguinal Ring RNA Sequencing Reveals Downregulation of Muscular Genes Related to Scrotal Hernia in Pigs.

Scrotal hernias (SH) are common congenital defects in commercial pigs, characterized by the presence of abdominal contents in the scrotal sac, leading to considerable production and animal welfare losses. Since the etiology of SH remains obscure, we aimed to identify the biological and genetic mechanisms involved in its occurrence through the whole transcriptome analysis of SH affected and unaffected pigs' inguinal rings. From the 22,452 genes annotated in the pig reference genome, 13,498 were expressed in the inguinal canal tissue. Of those, 703 genes were differentially expressed (DE, FDR < 0.05) between the two groups analyzed being, respectively, 209 genes upregulated and 494 downregulated in the SH-affected group. Thirty-seven significantly overrepresented GO terms related to SH were enriched, and the most relevant biological processes were muscular system, cell differentiation, sarcome reorganization, and myofibril assembly. The calcium signaling, hypertrophic cardiomyopathy, dilated cardiomyopathy, and cardiac muscle contraction were the major pathways possibly involved in the occurrence of the scrotal hernias. The expression profile of the DE genes was associated with the reduction of smooth muscle differentiation, followed by low calcium content in the cell, which could lead to a decreased apoptosis ratio and diminished muscle contraction of the inguinal canal region. We have demonstrated that genes involved with musculature are closely linked to the physiological imbalance predisposing to scrotal hernia. According to our study, the genes MYBPC1, BOK, SLC25A4, SLC8A3, DES, TPM2, MAP1CL3C, and FGF1 were considered strong candidates for future evaluation.

Identification of endogenous normalizing genes for expression studies in inguinal ring tissue for scrotal hernias in pigs.

The use of reference genes is required for relative quantification in gene expression analysis and since the stability of these genes could be variable depending on the experimental design, it has become indispensable to test the reliability of endogenous genes. Therefore, this study evaluated 10 reference candidate genes in two different experimental conditions in order to obtain stable genes to be used as reference in expression studies related to scrotal hernias in pigs. Two independent experiments were performed: one with 30 days-old MS115 pigs and the other with 60 days-old Landrace pigs. The inguinal ring/canal was collected, frozen and further submitted to real-time PCR analysis (qPCR). For the reference genes stability evaluation, four tools were used: GeNorm in the SLqPCR, BestKeeper, NormFinder and Comparative CT. A general ranking was generated using the BruteAggreg function of R environment. In this study, the RPL19 was one of the most reliable endogenous genes for both experiments. The breed/age effects influenced the expression stability of candidate reference genes evaluated in the inguinal ring of pigs. Therefore, this study reinforces the importance of evaluating the stability of several endogenous genes previous their use, since a consensual set of reference genes is not easily obtained. Here, two sets of genes are recommended: RPL19, RPL32 and H3F3A for 30-days MS115 and PPIA and RPL19 for the 60 days-old Landrace pigs. This is the first study using the inguinal ring tissue and the results can be useful as an indicative for other studies working with gene expression in this tissue.

Post-weaning piglets fed with different levels of fungal mycotoxins and spray-dried porcine plasma have improved weight gain, feed intake and reduced diarrhea incidence.

Mycotoxins are responsible for economic losses in the swine production industry, especially during post-weaning, when piglets are physiologically immature. Spray-dried porcine plasma (SDPP), added to pig diets, may help reduce losses due to mycotoxins. This work investigates the effects of SDPP in post-weaning piglets fed with diets containing natural contaminants or with more contaminants (co-contamination by mycotoxins). Fifty-six castrated weaned piglets were used in a randomized 2 (0 and 6% of SDPP) x 2 (natural contamination or co-contamination with mycotoxin) factorial design, with seven experimental units of two piglets each. The natural contaminants were 0.95 µg/kg aflatoxins +450 µg/kg fumonisins. The co-contaminated diet contained 300 µg/kg aflatoxins +8000 µg/kg fumonisins. Animals were fed 15 days with experimental diets. Feed intake, weight gain, feed efficiency, diarrhea incidence, and economic feasibility of SDPP treatement were evaluated in three periods of five days each. There was no interaction (P < 0.05) between mycotoxins levels and SDPP. Feed intake, weight gain and feed efficiency were higher (P < 0.05) in diets supplemented with SDPP. Animals fed with SDPP showed lower (P < 0.05) diarrhea incidence in the 1-10 day and 1-15 day periods. The experimental dose of mycotoxins reduced (P < 0.05) weight gain at 11-15 days. SDPP proved to be economical feasible over the total experimental period (1-15 days). Spray-dried plasma improved weight gain, feed intake and reduced diarrhea incidence in piglets post-weaning, but did not correlate with various levels of mycotoxins.