l-Arginine supplementation of gilts during early gestation modulates energy sensitive pathways in pig conceptuses.

Dietary l-arginine (ARG) supplementation has been studied as a nutritional strategy to improve reproductive performance of pregnant sows, since arginine is a conditionally essential amino acid. However, reports addressing the molecular mechanisms that mediate supplementation effects on embryos and fetuses development are still scarce. Therefore, we aimed to evaluate the effects of 1.0% ARG supplementation of commercial pregnant gilts on genes and proteins from energy metabolism and antioxidant defense pathways in embryos and fetuses. We also analyzed the global transcriptome profile of 25- and 35-day-old conceptuses. At Day 25, we observed a lower abundance of phospho-AMP-activated protein kinase (phospho-AMPK) protein and downregulation of oxidative phosphorylation system genes in ARG embryos. On the other hand, ARG fetuses showed greater expression of MLST8 and lower expression of MTOR genes, in addition to lower abundance of phospho-AMPK and phospho-mammalian target of rapamycin (phospho-mTOR) proteins. Transcriptome analysis at Day 35 did not present differentially expressed genes. For the antioxidant defense pathway, no differences were found between CON and ARG conceptuses, only trends. In general, supplementation of gilts with 1.0% ARG during early gestation affects energy sensitive pathways in 25- and 35-day conceptuses; however, no effects of supplementation were found on the antioxidative defense pathway in 25-day embryos.

Transcription Landscape of the Early Developmental Biology in Pigs.

Since pre- and postnatal development are programmed during early prenatal life, studies addressing the complete transcriptional landscape during organogenesis are needed. Therefore, we aimed to disentangle differentially expressed (DE) genes between fetuses (at 35 days old) and embryos (at 25 days old) through RNA-sequencing analysis using the pig as model. In total, 1705 genes were DE, including the top DE IBSP, COL6A6, HBE1, HBZ, HBB, and NEUROD6 genes, which are associated with developmental transition from embryos to fetuses, such as ossification, skeletal muscle development, extracellular matrix organization, cardiovascular system, erythrocyte differentiation, and neuronal system. In pathway analysis, embryonic development highlighted those mainly related to morphogenic signaling and cell interactions, which are crucial for transcriptional control during the establishment of the main organs in early prenatal development, while pathways related to myogenesis, neuronal development, and cardiac and striated muscle contraction were enriched for fetal development, according to the greater complexity of organs and body structures at this developmental stage. Our findings provide an exploratory and informative transcriptional landscape of pig organogenesis, which might contribute to further studies addressing specific developmental events in pigs and in other mammals.

Sex Determination Using RNA-Sequencing Analyses in Early Prenatal Pig Development.

Sexual dimorphism is a relevant factor in animal science, since it can affect the gene expression of economically important traits. Eventually, the interest in the prenatal phase in a transcriptome study may not comprise the period of development in which male and female conceptuses are phenotypically divergent. Therefore, it would be interesting if sex differentiation could be performed using transcriptome data, with no need for extra techniques. In this study, the sex of pig conceptuses (embryos at 25 days-old and fetuses at 35 days-old) was determined by reads counts per million (CPM) of Y chromosome-linked genes that were discrepant among samples. Thus, ten genes were used: DDX3Y, KDM5D, ZFY, EIF2S3Y, EIF1AY, LOC110255320, LOC110257894, LOC396706, LOC100625207, and LOC110255257. Conceptuses that presented reads CPM sum for these genes (ΣCPMchrY) greater than 400 were classified as males and those with ΣCPMchrY below 2 were classified as females. It was demonstrated that the sex identification can be performed at early stages of pig development from RNA-sequencing analysis of genes mapped on Y chromosome. Additionally, these results reinforce that sex determination is a mechanism conserved across mammals, highlighting the importance of using pigs as an animal model to study sex determination during human prenatal development.

Morphological and molecular differences in corpus luteum of pregnant sows from divergent genetic groups.

Comprehending mechanisms controlling corpus luteum (CL) angiogenesis and apoptosis in pregnant sows is essential to understand the physiological role of these processes in CL function, progesterone production and consequently in conceptus development and prenatal mortality. CL from 54 sows from two genetic groups, a commercial line (COM) and the local Piau breed (LPB), were obtained for gene expression (n = 3 COM; n = 6 LPB), histological and protein analysis (n = 3 COM; n = 3 LPB), divided in six gestational ages (seven, 15, 30, 45, 60 and 90 days). We observed differences between gestational ages in CL morphology, in which the average number of blood vessels/capillaries at 90-days was greater than at the seventh day by Tukey test. RT-qPCR analysis revealed that apoptotic genes (BAX, BCL2 and CASP3) were differentially expressed between genetic groups and gestational ages in each group. Angiogenesis genes also presented differences between genetic groups (ANGPT1) and gestational ages (MMP9, VEGFA and ANGPT1). No differences in protein abundance of steroidogenic enzymes (CYP11A1 and HSD3B1) were observed. Our findings indicate that despite the differences in gene expression, differences in corpus luteum vascularization were observed only across gestational ages, with no dissimilarities between genetic groups.