[The impact of phenolic compounds on pig fat deposition].

The quantity and distribution of fat deposits are crucial factors that impact the quality of pork. Recent studies have indicated that the utilization of natural ingredients plays a significant role in decreasing subcutaneous and visceral fat deposits, as well as enhancing intramuscular fat. Moreover, natural products possess several advantages including being environmentally friendly, safe, free of additives, and leaving no residue. Phenolic compounds derived from fruits, vegetables and herbs constitute of vital component of these natural ingredients. This article examines the influence of phenolic compounds on pig fat deposition, aiming to provide guidance on the utilization of such compounds to regulate fat deposition and enhance pork quality.

Induction of lung progenitor cell-like organoids by porcine pluripotent stem cells.

Organoids are in vitro 3D models that are generated using stem cells to study organ development and regeneration. Despite the extensive research on lung organoids, there is limited information on pig lung cell generation or development. Here, we identified five epithelial cell types along with their characteristic markers using scRNA-seq. Additionally, we found that NKX2.1 and FOXA2 acted as the crucial core transcription factors in porcine lung development. The presence of SOX9/SOX2 double-positive cells was identified as a key marker for lung progenitor cells. The Monocle algorithm was used to create a pseudo-temporal differentiation trajectory of epithelial cells, leading to the identification of signaling pathways related to porcine lung development. Moreover, we established the differentiation method from porcine pluripotent stem cells (pPSCs) to SOX17+ FOXA2+ definitive endoderm (DE) and NKX2.1+ FOXA2+ CDX2- anterior foregut endoderm (AFE). The AFE is further differentiated into lung organoids while closely monitoring the differentiation process. We showed that NKX2.1 overexpression facilitated the induction of lung organoids and supported subsequent lung differentiation and maturation. This model offers valuable insights into studying the interaction patterns between cells and the signaling pathways during the development of the porcine lung.

Sophoridine Counteracts Obesity via Src-Mediated Inhibition of VEGFR Expression and PI3K/AKT Phosphorylation.

Sophoridine (SRP) is a natural quinolizidine alkaloid found in many traditional Chinese herbs, though its effect on adipose tissue is unclear. We improved serum lipid levels by administering SRP by gavage in high-fat diet (HFD)-fed C57BL/6 mice. After 11 weeks, SRP supplementation significantly reduced body weight gain and improved glucose homeostasis, while reducing subcutaneous fat and liver weight. SRP also inhibited cell proliferation and differentiation of 3T3-L1 cells. Proteomics analysis revealed that SRP inhibits adipocyte differentiation by interacting with Src, thereby suppressing vascular endothelial growth factor receptor 2 (VEGFR2) expression and PI3K/AKT phosphorylation. This study provides an empirical basis for the treatment of obesity with small molecules.

miR-103-3p Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2.

Skeletal muscle, a vital and intricate organ, plays a pivotal role in maintaining overall body metabolism, facilitating movement, and supporting normal daily activities. An accumulating body of evidence suggests that microRNA (miRNA) holds a crucial role in orchestrating skeletal muscle growth. Therefore, the primary aim of this study was to investigate the influence of miR-103-3p on myogenesis. In our study, the overexpression of miR-103-3p was found to stimulate proliferation while suppressing differentiation in C2C12 myoblasts. Conversely, the inhibition of miR-103-3p expression yielded contrasting effects. Through bioinformatics analysis, potential binding sites of miR-103-3p with the 3'UTR region of BTG anti-proliferative factor 2 (BTG2) were predicted. Subsequently, dual luciferase assays conclusively demonstrated BTG2 as the direct target gene of miR-103-3p. Further investigation into the role of BTG2 in C2C12 myoblasts unveiled that its overexpression impeded proliferation and encouraged differentiation in these cells. Notably, co-transfection experiments showcased that the overexpression of BTG2 could counteract the effects induced by miR-103-3p. In summary, our findings elucidate that miR-103-3p promotes proliferation while inhibiting differentiation in C2C12 myoblasts by targeting BTG2.

[Porcine skeletal muscle development regulated by MicroRNA: a review].

The growth and development of skeletal muscle is an important factor affecting pork production and quality, which is elaborately regulated by many genetic and nutritional factors. MicroRNA (miRNA) is a non-coding RNA with a length of about 22 nt, which binds to the 3'UTR sequence of the mRNA of the target genes, and consequently regulates its post-transcriptional expression level. In recent years, a large number of studies have shown that miRNAs are involved in various life processes such as growth and development, reproduction, and diseases. The role of miRNAs in the regulation of porcine skeletal muscle development was reviewed, with the hope to provide a reference for the genetic improvement of pigs.

Genome-Wide Association Study on Reproductive Traits Using Imputation-Based Whole-Genome Sequence Data in Yorkshire Pigs.

Reproductive traits have a key impact on production efficiency in the pig industry. It is necessary to identify the genetic structure of potential genes that influence reproductive traits. In this study, a genome-wide association study (GWAS) based on chip and imputed data of five reproductive traits, namely, total number born (TNB), number born alive (NBA), litter birth weight (LBW), gestation length (GL), and number of weaned (NW), was performed in Yorkshire pigs. In total, 272 of 2844 pigs with reproductive records were genotyped using KPS Porcine Breeding SNP Chips, and then chip data were imputed to sequencing data using two online software programs: the Pig Haplotype Reference Panel (PHARP v2) and Swine Imputation Server (SWIM 1.0). After quality control, we performed GWAS based on chip data and the two different imputation databases by using fixed and random model circulating probability unification (FarmCPU) models. We discovered 71 genome-wide significant SNPs and 25 potential candidate genes (e.g., SMAD4, RPS6KA2, CAMK2A, NDST1, and ADCY5). Functional enrichment analysis revealed that these genes are mainly enriched in the calcium signaling pathway, ovarian steroidogenesis, and GnRH signaling pathways. In conclusion, our results help to clarify the genetic basis of porcine reproductive traits and provide molecular markers for genomic selection in pig breeding.

Dietary grape pomace extract supplementation improved meat quality, antioxidant capacity, and immune performance in finishing pigs.

In pig production, reducing production costs and improving immunity are important. Grape pomace, a good agricultural by-product, has been thrown away as food waste for a long time. Recently, we found that it could be used as a new source of pig feed. We investigated the effect of grape pomace on inflammation, gut barrier function, meat quality, and growth performance in finishing pigs. Our results indicated that treatment samples showed a significant decrease in water loss, IL-1ß, DAO, ROS, and MDA content (p < 0.05). IgA, IgG, IgM, CAT, T-AOC, SOD, and IFN-γ significantly increased compared with those in control samples (p < 0.05). Meanwhile, the relative mRNA expression of the tight junction protein occludin showed a significant difference (p < 0.05). Analysis of metagenomic sequencing indicated that grape pomace significantly decreased the relative abundance of Treponema and Streptococcus (p < 0.05). In summary, our results demonstrated that grape pomace could improve meat quality, alleviate inflammation, and decrease oxidative stress.

Transcriptome, proteome and metabolome analysis provide insights on fat deposition and meat quality in pig.

Intramuscular fat (IMF) content, which is an important determinant of meat quality characteristics such as tenderness, juiciness and flavor, has long been a research hotspot. Chinese local pig breeds are famous for their excellent meat quality which is mainly reflected in the high IMF content, strong hydraulic system and et al. However, there are few analysis of meat quality by omics methods. In our study, we identified 12 different fatty acids, 6 different amino acids, 1,262 differentially expression genes (DEGs), 140 differentially abundant proteins (DAPs) and 169 differentially accumulated metabolites (DAMs) (p < 0.05) with metabolome, transcriptome, and proteome. It has been found that DEGs, DAPs and DAMs were enriched in the Wnt signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway, and Ras signaling pathway which were related to meat quality. Moreover, our Weighted genes co-expression network construction (WGCNA) showed RapGEF1 was the key gene related to IMF content and the RT-qPCR analysis was used to perform validation of the significant genes. In summary, our study provided both fundamental data and new insights to further uncover the secret of pig IMF content.

Comparative analysis of porcine iPSCs derived from Sertoli cells and fibroblasts.

Porcine embryonic fibroblasts (PEFs) can be directly reprogrammed into porcine induced pluripotent stem cells (piPSCs). However, the reprogramming process is generally lengthy and inefficient. Here, we established a fast and efficient induction system of piPSCs from porcine Sertoli cells (SCs) via forced expression of pig Yamanaka factors. The alkaline phosphatase (AP)-positive colonies from SCs developed on Day 3 after lentivirus infection, and were expanded and then picked up on Day 7, whereas reprogramming process from PEFs did not show any colonies in the same period. The picked piPSCs strongly expressed pluripotent genes, had the differentiation capacity to three germ layers, and could be also induced into primordial germ cell-like cells. Screening for transcription factor combinations showed that POU class 5 homeobox 1 (OCT4) is the core factor for AP-positive colony formation, and two factors (OCT4 and c-MYC) could successfully reprogram SCs into piPSCs. We then compared the RNA-sequencing data of piPSCs derived from SCs and PEFs, and found that the most significant difference was the activation of Transforming Growth Factor ß signaling pathway. We also compared the RNA levels of SCs and PEFs, and found that SCs exhibited higher Wnt signaling activity and Bone Morphogenetic Protein 4 expression than PEFs, which might be correlated with higher cell proliferation rate and reprogramming efficiency. In summary, the data demonstrated that starting cell sources of piPSCs significantly affect reprogramming dynamics and SCs could serve as cell sources for efficient reprogramming.

Ablation of Tas1r1 Reduces Lipid Accumulation Through Reducing the de Novo Lipid Synthesis and Improving Lipid Catabolism in Mice.

Amino acid sensing plays an important role in regulating lipid metabolism by sensing amino acid nutrient disturbance. T1R1 (umami taste receptor, type 1, member 1) is a membrane G protein-coupled receptor that senses amino acids. Tas1r1-knockout (KO) mice were used to explore the function of umami receptors in lipid metabolism. Compared with wild-type (WT) mice, Tas1r1-KO mice showed decreased fat mass (P < 0.05) and adipocyte size, lower liver triglyceride (7.835 ± 0.809 vs 12.463 ± 0.916 mg/g WT, P = 0.013) and total cholesterol levels (0.542 ± 0.109 vs 1.472 ± 0.044 mmol/g WT, P < 0.001), and reduced lipogenesis gene expressions in adipose and liver tissues. Targeted liver amino acid metabolomics showed that the amino acid content of Tas1r1-KO mice was significantly decreased, which was consistent with the branched-chain ketoacid dehydrogenase protein levels. Proteomics analysis showed that the upregulated proteins were enriched in lipid and steroid metabolism pathways, and parallel reaction monitoring results illustrated that Tas1r1 ablation promoted lipid catabolism through oxysterol 7 α-hydroxylase and insulin-like growth factor binding protein 2. In summary, Tas1r1 disruption in mice could reduce lipid accumulation by reducing de novo lipid synthesis and improving lipid catabolism.

COPS3 AS lncRNA enhances myogenic differentiation and maintains fast-type myotube phenotype.

Long non-coding RNAs (lncRNAs) play essential roles in myogenesis. Here, we identified a novel long non-coding RNA, named COPS3 AS lncRNA (COP9 signalosome complex subunit 3 antisense lncRNA), which was transcribed from the mouse COPS3 gene antisense strand and highly expressed in glycolytic muscle fibers. Functionally, COPS3 AS lncRNA knockdown inhibited myogenic differentiation in myoblasts, whereas its overexpression promoted the process. Moreover, COPS3 AS lncRNA maintained the fast-twitch myotubes phenotype. Mechanistically, although COPS3 AS lncRNA did not form AS lncRNA/mRNA dimer with COPS3 mRNA, it as a competing endogenous RNA (ceRNA) to sponge miR-762, promoted myogenic differentiation and Fast-MyHC expression by modulating miR-762 target gene myogenic differentiation 1 (MyoD1). Taken together, COPS3 AS lncRNA is a key candidate regulator of myogenesis and fast-MyHC myotubes specification by miR-762/MyoD signalling axis.

Single-cell RNA-seq analysis of testicular somatic cell development in pigs.

Spermatogenesis is the process by which diploid male germ cells propagate and differentiate into haploid flagellated spermatozoa. This highly complicated process is dependent on testicular somatic cells maturation. While the role of these somatic cells in spermatogenesis is relatively well established, knowledge about their development and maturation, particularly at the molecular level, is limited. In this study, we profiled the testicular single-cell transcriptomes of Guanzhong black pigs at the age of 7, 30, 60, 90, and 150 days. Five types of Sertoli cells, five types of Leydig cells, and four types of peritubular myoid cells were identified. Histological analysis revealed the changes in proliferation levels and marker gene expressions, and the prion-like protein gene (PRND) was identified as a novel marker for Sertoli cells. Additionally, integrated analyses of porcine and human datasets revealed similarities between human and pig testicular somatic cells. Overall, the data obtained in this study contribute to the understanding of testicular development in pigs as a model species.

Porcine Endogenous Retroviruses: Quantification of the Viral Copy Number for the Four Miniature Pig Breeds in China.

Domestic pigs has served not only as one of the most important economy livestock but also as ideal organ-source animals owing to similarity in anatomy, physiology, and organ size to humans. Howerer, the barrier of the cross-species transmission risk of porcine endogenous retrovirus (PERVs) blocked the pig-to-human xenotransplantation. PERVs are integrated into pigs' genomes and cannot be eliminated by designated or specified pathogen-free breeding. PERVs are an important biosafety issue in xenotransplantation because they can be released from normal pig cells and infect human cells in vitro under certain conditions. Screening and analyzing the presence of PERVs in pig genome will provide essential parameters for pig breed sources. In China, four miniature pig breeds, such as Guizhou miniature pig (GZ), Bama miniature pig (BM), Wuzhishan miniature pig (WZS), and Juema miniature pig (JM), were the main experimental miniature pig breeds, which were widely used. In this study, PCR was performed to amplify env-A, env-B, and env-C for all individuals from the four breeds. The results revealed that PERV env-A and env-B were detected in all individuals and the lowest ratios of PERV env-C was 17.6% (3/17) in the GZ breed. Then, PERV pol and GAPDH were detected using the droplet digital PCR (ddPCR) method. As the reference of GAPDH copy number, the copy numbers of PERVs were at the median of 12, 16, 14, and 16 in the four miniature pig breeds (GZ, BM, WZS, and JM), respectively. Furthermore, the copy number of the PERV pol gene in many organs from the GZ breed was analyzed using ddPCR. The copy numbers of PERV pol gene were at the median of 7 copies, 8 copies, 8 copies, 11 copies, 5 copies, 6 copies, and 7 copies in heart, liver, spleen, lung, kidney, muscle, and skin, and the maximum number was 11 copies in the lung. The minimum number was 5 copies in the kidney as the reference of GAPDH. These data suggest that GZ breed has the lower PERVs copy number in the genome, and may be an ideal organ-source miniature pig breed for the study of the pig-to-human xenotransplantation.

Hydroxytyrosol effectively improves the quality of pig sperm at 17 °C.

Artificial insemination (AI) is a proven breeding technology which has been widely used in pig reproduction. Low temperature can cause very serious damage to pig sperm below 15 °C and the situation is even more serious at lower temperature. Besides, the preservation of pig sperm is mainly carried out at 17 °C because of its outstanding performance in pig reproduction. However, the accumulation of a large amount of reactive oxygen species (ROS) during the preservation process is the main reason for the deterioration of sperm quality. In our research, by adding different concentrations of hydroxytyrosol to the diluent during the storage of pig sperm at 17 °C, we compared them with the traditional diluent to study the sperm motility, the cumulative amount of ROS, the extent of sperm membrane damage, the sperm acrosome integrity, the sperm DNA damage and the activity of antioxidant enzymes (CAT, T-AOC, SOD, GSH-PX, MDA) to evaluate the effect of hydroxytyrosol on the sperm quality during storage. We used proteomics sequencing technology to monitor difference in sperm protein between the control samples and the addition of 120 µmol/L hydroxytyrosol samples (optimum concentration) after three days storage. Ultimately, we selected the control samples and the addition of 120 µmol/L hydroxytyrosol samples to test the effect of AI. The results of our research showed that during storage of pig sperm at 17 °C, the sperm quality and antioxidant capacity of the hydroxytyrosol-treated samples significantly improved (HT 120 µmol/L) (P < 0.05). Proteomics sequencing analysis proved that the addition of 120 µmol/L hydroxytyrosol treatment samples had potential value in improving sperm quality. The significant increase in sow pregnancy rate and piglet birth weight proved that hydroxytyrosol had important practical value in pig reproduction. Based on our results, we demonstrated that the addition of hydroxytyrosol to the diluent could improve the quality of pig sperm and the efficiency of AI.

Single-cell RNA-sequencing reveals the dynamic process and novel markers in porcine spermatogenesis.

BACKGROUND: Spermatogenesis is the process by which male gametes are formed from spermatogonial stem cells and it is essential for the reliable transmission of genetic information between generations. To date, the dynamic transcriptional changes of defined populations of male germ cells in pigs have not been reported. RESULTS: To characterize the atlas of porcine spermatogenesis, we profiled the transcriptomes of ~ 16,966 testicular cells from a 150-day-old pig testis through single-cell RNA-sequencing (scRNA-seq). The scRNA-seq analysis identified spermatogonia, spermatocytes, spermatids and three somatic cell types in porcine testes. The functional enrichment analysis demonstrated that these cell types played diverse roles in porcine spermatogenesis. The accuracy of the defined porcine germ cell types was further validated by comparing the data from scRNA-seq with those from bulk RNA-seq. Since we delineated four distinct spermatogonial subsets, we further identified CD99 and PODXL2 as novel cell surface markers for undifferentiated and differentiating spermatogonia, respectively. CONCLUSIONS: The present study has for the first time analyzed the transcriptome of male germ cells and somatic cells in porcine testes through scRNA-seq. Four subsets of spermatogonia were identified and two novel cell surface markers were discovered, which would be helpful for studies on spermatogonial differentiation in pigs. The datasets offer valuable information on porcine spermatogenesis, and pave the way for identification of key molecular markers involved in development of male germ cells.

Transcriptome-wide Dynamics of m6A mRNA Methylation During Porcine Spermatogenesis.

Spermatogenesis is a continual process that occurs in the testes, in which diploid spermatogonial stem cells (SSCs) differentiate and generate haploid spermatozoa. This highly efficient and intricate process is orchestrated at multiple levels. N6-methyladenosine (m6A), an epigenetic modification prevalent in mRNAs, is implicated in transcriptional regulation during spermatogenesis. However, the dynamics of m6A modification in non-rodent mammalian species remains unclear. Here we systematically investigated the profile and role of m6A during spermatogenesis in pigs. By analyzing the transcriptomic distribution of m6A in spermatogonia, spermatocytes, and round spermatids, we identified a globally conserved m6A pattern between porcine and murine genes with spermatogenic function. We found that m6A was enriched in a group of genes that specifically encode the metabolic enzymes and regulators. In addition, transcriptomes in porcine male germ cells could be subjected to the m6A modification. Our data showed that m6A played the regulatory roles during spermatogenesis in pigs, which is similar to that in mice. Illustrations of this point were three genes (SETDB1, FOXO1, and FOXO3) that were crucial to the determination of the fate of SSCs. To the best of our knowledge, this study has for the first time uncovered the expression profile and role of m6A during spermatogenesis in large animals and contributes to insights into the intricate transcriptional regulation underlying the lifelong male fertility in non-rodent mammalian species.

MiR-99b-5p Attenuates Adipogenesis by Targeting SCD1 and Lpin1 in 3T3-L1 Cells.

The number and distribution of adipocytes directly affect the quality of livestock meat products. The analysis of the adipogenesis mechanism is the basis for improving meat quality. The formation of adipocytes is regulated by many factors, including a class of endogenous small RNAs, named microRNA (miRNA). Previous studies have shown that miRNAs could affect adipogenesis by post-transcriptional regulation of target genes. In our study, a decreased miR-99b-5p expression level was found in the adipose tissue of obese mice. Overexpression of miR-99b-5p could increase cell proliferation by promoting the cell cycle while inhibiting cell differentiation. In addition, interference with miR-99b-5p obtained the opposite result. Furthermore, the proteomics sequencing analysis screened 1154 differentially expressed proteins, which are closely related to adipocyte differentiation and fatty acid metabolism. In addition, the results of the dual-luciferase test showed that miR-99b-5p can directly target the proteins SCD1 and Lpin1 with significantly different expression levels in proteomic sequencing. Then, this result was verified at the level of mRNA and protein in a further study. Collectively, these results suggested that miR-99b-5p may be a target for improving meat quality.

Adipose-specific BMP and activin membrane-bound inhibitor (BAMBI) deletion promotes adipogenesis by accelerating ROS production.

With the improvement of people's living standards, the number of obese patients has also grown rapidly. It is reported that the level of oxidative stress in obese patients has significantly increased, mainly caused by the increase in reactive oxygen species (ROS) levels in adipose tissue. Studies have shown that the use of siRNA to interfere with bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) expression could promote adipocyte differentiation, and under hypoxic conditions, BAMBI could act as a regulator of HIF1α to regulate the polarity damage of epithelial cells. In view of these results, we speculated that BAMBI may regulate adipogenesis by regulating the level of ROS. In this study, we generated adipose-specific BAMBI knockout mice (BAMBI AKO) and found that compared with control mice, BAMBI AKO mice showed obesity when fed with high-fat diet, accompanied by insulin resistance, glucose intolerance, hypercholesterolemia, and increased inflammation in adipose tissue. Interestingly, adipose-specific deficiency of BAMBI could cause an increase in the expression level of Nox4, thereby promoting ROS production in cytoplasm and mitochondria and the DNA-binding activity of C/EBPß and ultimately promoting adipogenesis. Consistently, our findings indicated that BAMBI may be a reactive oxygen regulator to affect adipogenesis, thereby controlling obesity and metabolic syndrome.

Boar sperm quality and oxidative status as affected by rosmarinic acid at 17 °C.

Peroxidation damage induces sublethal injury to boar sperm during preservation. Rosmarinic acid (RA) has already been verified to efficiently protect cells from oxidant-induced injury and to produce significant effect on cryopreservation of semen. Through our experiments, we aim at investigating whether RA has a positive effect on the preservation of pig semen at room temperature. The semen collected from sexually mature Large White boars were preserved at 17 °C in Beltsville thawing solution (BTS) supplied. The boar sperm were exposed to 0, 25, 50, 75, 100, 125 and 150 µM RA in vitro and the sperm functions were examined. The sperm motility, the acrosome and plasma membrane integrity, the catalase activity (CAT), the total antioxidative capacity (T-AOC) activity and the malondialdehyde content (MDA) were examined at 0, 1, 3 and 5 days. The BTS diluent containing RA improved the sperm quality during the process of liquid preservation compared with the control treatment. After 5 days of liquid preservation, the addition of RA at 100 µM produced an optimal effect on the survival time as well as on the maintenance of motility, acrosome and plasma membrane integrity; T-AOC activity; CAT activity; and the MDA content. Besides, our results in the reproductive experiments showed that the addition of RA at 100 µM to the BTS diluent increased the pregnancy rate. These results suggest that the proper concentration of RA in boar semen extenders possibly improves the artificial insemination efficiency by reducing the sperm damage and the subsequent dysfunction during liquid preservation in swine production systems.