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Abstract The Wannan black pig is a superior local breed in Anhui province, renowned for its exceptional meat quality and remarkable adaptability to various environmental conditions. Semen, being a crucial indicator of male sexual maturity and fertility, significantly influences the performance of breeding boars. The molecular basis for comprehending the fecundity of boars in practical production lies in understanding the disparities in sperm proteins among boars of varying ages. In this investigation, spermatozoa from three one-year-old and three seven-year-old Wannan black pigs were individually chosen. Employing a tandem mass tag (TMT)-based quantitative proteomics approach, a total of 4050 proteins were identified, out of which 130 proteins exhibited significant differences between the two groups. GO enrichment analysis revealed that these proteins primarily participated in energy metabolism, spermatogenesis, fertilization, and reproduction. KEGG enrichment analysis demonstrated that the differential proteins predominantly resided within the ribosome pathway. A protein-protein interaction (PPI) network was constructed to identify core proteins such as Small ribosomal subunit protein uS7 (RPS5). Ultimately, parallel reaction monitoring (PRM) was conducted on the selected differential proteins to validate result accuracy. The findings of this study establish a foundation for elucidating the molecular mechanism underlying variations in spermatozoa protein levels among Wannan Black Pig with different age.

Abstract VPS28 (vacuolar protein sorting 28) is a subunit of the endosomal sorting complexes required for transport (ESCRTs) and is involved in ubiquitination. Ubiquitination is a critical system for protein degradation in eukaryotes. Considering the recent findings on the role of ubiquitination in the regulation of lipid metabolism, we hypothesized that VPS28 might affect the expression of genes involved in milk fat synthesis. To test this hypothesis, we modulated VPS28 expression in the bovine mammary epithelial cell line (MAC-T) and measured the effects on triglyceride (TG) synthesis using lentivirus-mediated techniques. The results showed that VPS28 knockdown significantly upregulated the levels of the fatty acid transporter CD36 molecule (CD36) and adipose differentiation-related protein (ADFP), leading to increased TG and fatty acid production, along with elevated ubiquitin (UB) levels, while reducing proteasome activity. In contrast, VPS28 overexpression increased CD36 levels while not significantly affecting ADFP or TG levels, with a trend toward reduced lipid droplets and increased UB expression and proteasome activity. In addition, inhibition of the ubiquitin-proteasome system and the endosomal-lysosomal pathway using epoxomicin and chloroquine, respectively, further increased CD36, ADFP, and TG levels, thereby enhancing cell viability. These in vitro findings were validated in vivo in a mouse model, where VPS28 knockdown increased mammary CD36, ADFP, UB expression, TG content, and lipid droplets without pathological changes in mammary tissue or blood TG alterations. These results confirm the pivotal role of VPS28 in regulating TG synthesis via the ubiquitination pathway, offering novel insights into the molecular mechanisms of milk fat production in a bovine cell model.

According to previous studies, the quality and fertilization rate of fresh sperm from boars of different ages were significantly different. However, the difference of freeze–thaw sperm quality and fertility in boars of different ages is unclear. In this study, boars of a Chinese native breed were assigned into two groups. Each group consisted of five boars aged aged either 2–3 years (young boars = YB) or 5–6 years (aging boars = AB) A total of 60 ejaculates for each group were collected and cryopreserved. Semen quality and in vitro fertility of post-thaw sperm was evaluated. The results showed that the concentration and motility of fresh sperm collected from AB were similar to YB, but their semen volume was higher than that in YB (p

The quality of frozen pork is adversely affected upon thawing. In this study, the influence of various thawing methods for frozen pork, including cold water (18 °C), room temperature (18 °C), and refrigeration (4 °C) thawing, on physicochemical and nutrient substances were examined. The pork samples (a Chinese local breed: Anqing six-end-white pigs), which were thawed through the above conditions, were compared with controls (fresh porcine longissimus dorsi). Analyses were carried out to determine porcine longissimus dorsi shear force, pH value, crude protein content, antioxidant capacity, amino acid content, and fatty acid content. The results indicated that the shear force, pH value, crude protein content, total antioxidant capacity (T-AOC), and total superoxide dismutase (T-SOD) content of the porcine longissimus dorsi muscle significantly decreased via the three thawing methods compared with the control group (p < 0.05). However, malondialdehyde (MDA) content, intramuscular fat content, inosinate and cholesterol content, essential amino acid content, and umami amino acid proportion in the cold thawing group were not significantly different from the control group (p > 0.05), but there were significant differences between the other two groups. The MDA content of the air thawing and hydrostatic thawing groups significantly increased compared with the control group (p < 0.05), with it being 42.6% and 50.8% higher than the control group, respectively. In addition, the monounsaturated fatty acid content in the pork subjected to the three thawing methods significantly increased compared with the control group (p < 0.05), and the monounsaturated fatty acid content after cold thawing and hydrostatic thawing increased by 18.2% and 21.6%, respectively. In conclusion, refrigeration had less influence on the quality of the Anqing six-end-white pork and was the most suitable thawing method. This study provides a theoretical reference for frozen pork preservation for improving food quality and availing its economic benefits.

Interferon regulatory factor 8 (IRF8) is a key regulator of innate immune receptor signaling that resists pathogen invasion by regulating cell growth and differentiation. Porcine epidemic diarrhea virus (PEDV) targets the intestine and damages the mucosal barrier. However, whether IRF8 regulates PEDV replication remains unclear. We revealed that PEDV infection activated IRF8 expression. Moreover, IRF8 deletion drastically promoted PEDV replication and invasion, increasing the virus copies and titers. Hypomethylation enrichment of activating protein (AP)-2α was significantly negatively correlated with high IRF8 expression, and AP-2α directly targeted the IRF8 promoter to regulate PEDV replication. Furthermore, IRF8 overexpression decreased the cellular reactive oxygen species levels and mitochondrial membrane potential and increased the antioxidant enzyme activities to alleviate PEDV-induced oxidative damage. IRF8 overexpression suppressed apoptotic gene expression, thereby inhibiting apoptosis in response to PEDV stimulation. Taken together, this study demonstrates that AP-2α is involved in PEDV-induced epigenetic modification of IRF8 to reduce cell apoptosis and oxidative stress and facilitate host resistance to PEDV in the intestinal epithelium.

Pubertal genetic variations between the indigenous Chinese Wanyue Black pig breed and the imported Yorkshire breed significantly impact their reproductive capacity. In order to identify the differentially expressed genes, gene networks, and metabolic pathways in ovary transcriptome of gilts, the serum hormone levels were analyzed by ELISA, and RNA-seq was performed to analyze ovarian genes. Our results reveal higher estradiol (E2) levels in Wanyue black gilts compared to Yorkshire gilts, while Yorkshire gilts exhibit elevated progesterone (P4) and GnRH levels. We identified a total of 154 differentially expressed genes (DEGs), with 87 up-regulated and 67 down-regulated genes in the Wanyue black gilts ovaries compared to the Yorkshire gilts. GO enrichment analysis unveiled the participation of DEGs in processes such as “Reproduction”, “Reproductive system development”, and “Ovarian follicle development”. Moreover, KEGG enrichment analysis revealed the involvement of DEGs in multiple signaling pathways associated with hormone biosynthesis and puberty, encompassing “Steroid hormone biosynthesis”, “Estrogen signaling pathway”, and “Prolactin signaling pathway”. The subsequent bioinformatics analysis identified nine functional genes that potentially contribute to the disparity in ovaries between Wanyue black gilts and Yorkshire gilts. This study offers significant insights into the endocrine and genetic aspects of pubertal development in gilts.

MicroRNAs (miRNAs) are small, noncoding RNAs that play a crucial role in the complex and dynamic network that regulates the apoptosis of porcine ovarian granulosa cells (POGCs). Resveratrol (RSV) is a nonflavonoid polyphenol compound that is involved in follicular development and ovulation. In previous study, we established a model of RSV treatment of POGCs, confirming the regulatory effect of RSV in POGCs. To investigate the miRNA-level effects of RSV on POGCs to reveal differentially expressed miRNAs, a control group (n = 3, 0 μM RSV group), a low RSV group (n = 3, 50 μM RSV group), and a high RSV group (n = 3, 100 μM RSV group) were created for small RNA-seq. In total, 113 differentially expressed miRNAs (DE-miRNAs) were identified, and a RT-qPCR analysis showed a correlation with the sequencing data. Functional annotation analysis revealed that DE-miRNAs in the LOW vs. CON group may be involved in cell development, proliferation, and apoptosis. In the HIGH vs. CON group, RSV functions were associated with metabolic processes and responses to stimuli, while the pathways were related to PI3K24, Akt, Wnt, and apoptosis. In addition, we constructed miRNA-mRNA networks related to Apoptosis and Metabolism. Then, ssc-miR-34a and ssc-miR-143-5p were selected as key miRNAs. In conclusion, this study provided an improved understanding of effects of RSV on POGCs apoptosis through the miRNA modulations. The results suggest that RSV may promote POGCs apoptosis by stimulating the miRNA expressions and provided a better understanding of the role of miRNAs combined with RSV in ovarian granulosa cell development in pigs.

Background: Drosophila ubiquitin carboxy-terminal hydrolase L5 (Uch-L5) functions as a critical component of the 26S proteasome to mediate degradation of polyubiquitinated proteins. It was recently shown to modulate tissue/organ development by targeting the Smoothened protein in the hedgehog pathway. However, whether it plays a role in controlling organismal immune response remains largely unknown. Methods: Reverse transcription plus quantitative polymerase chain reaction (RT-qPCR), dual-luciferase, and Western blot assays were used to explore the potential function of Uch-L5 in the innate immune regulation in cultured Drosophila S2 cells. Further genetic manipulations and bacterial infections were conducted to confirm the findings in vivo. Results: Silencing of Uch-L5 antagonizes the immune deficiency (IMD) but not the Toll innate immune signaling both in vitro and in vivo. Moreover, Uch-L5 positively contributes to the Drosophila innate immune response via its N-terminal Uch domain, which is the catalytical triad executing its deubiquitinase activity. Conclusions: Our studies shed light on a novel function of the deubiquitinase Uch-L5 in governing the anti-microbial defense in Drosophila.

Resveratrol (RES) is a non-flavonoid polyphenol compound that can be involved in follicular development and ovulation. However, the mechanism by which resveratrol regulates the apoptosis of porcine ovarian granulosa cells (POGCs) through long non-coding RNA (lncRNA) is poorly understood. We generated POGCs models of different doses of RES (0, 25, 50, 75, and 100 μM). It was observed that the cell viability was the highest in the 50 μM group, and the highest apoptosis rates were recorded in the 100 μM group. Therefore, a control group (n = 3, 0 μM RES group), a low RES group (n = 3, 50 μM RES group), and a high RES group (n = 3, 100 μM RES group) of POGCs were created for next RNA sequencing. Gene Ontology (GO) indicated that differentially expressed lncRNAs associated with apoptotic process were highly enriched. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of lncRNA target genes found that the Wnt signaling pathway and PI3K-Akt signaling pathway were both enriched. Furthermore, we constructed lncRNA-mRNA networks related to Metabolic and Cell Apoptosis, respectively. In the networks, five key-lncRNAs were screened, which may play a significant role in the process of POGCs metabolism and apoptosis. Furthermore, we focused on the function of a lnc-GAM (lncRNA associated with Granulosa cells Apoptosis and Metabolism) and verified that lnc-GAM could influence cell apoptosis in POGCs development by affecting the mRNA expression of apoptosis-related markers, and also affects the secretion of steroid hormones and related genes expression in POGCs cultured in vitro. Our study provides seminal data and important new insights into the regulation of reproductive mechanisms in porcine and other female mammals.

Background Previous studies have proven that bag-of-marbles (bam) plays a pivotal role in promoting early germ cell differentiation in Drosophila ovary. However, whether it functions in regulating the metabolic state of the host remains largely unknown. Methods We utilized GC-MS, qPCR, and some classical kits to examine various metabolic profiles and gut microbial composition in bam loss-of-function mutants and age-paired controls. We performed genetic manipulations to explore the tissue/organ-specific role of bam in regulating energy metabolism in Drosophila. The DSS-induced mouse colitis was generated to identify the role of Gm114, the mammalian homolog of bam, in modulating intestinal homeostasis. Results We show that loss of bam leads to an increased storage of energy in Drosophila. Silence of bam in intestines results in commensal microbial dysbiosis and metabolic dysfunction of the host. Moreover, recovery of bam expression in guts almost rescues the obese phenotype in bam loss-of-function mutants. Further examinations of mammalian Gm114 imply a similar biological function in regulating the intestinal homeostasis and energy storage with its Drosophila homolog bam. Conclusion Our studies uncover a novel biological function of bam/Gm114 in regulating the host lipid homeostasis.

Aspirin, also known as acetylsalicylic acid, is widely consumed as a pain reliever and an anti-inflammatory as well as anti-platelet agent. Recently, our studies using the animal model of Drosophila demonstrated that the dietary supplementation of aspirin renovates age-onset intestinal dysfunction and delays organismal aging. Nevertheless, it remains probable that aspirin plays functional roles in other biological activities, for instance antiviral defense reactions. Intriguingly, we observed that the replications of several types of viruses were drastically antagonized in Drosophila macrophage-like S2 cells with the addition of aspirin. Further in vivo experimental approaches illustrate that adult flies consuming aspirin harbor higher resistances to viral infections with respect to flies without aspirin treatment. Mechanistically, aspirin positively contributes to the Drosophila antiviral defense largely through mediating the STING (stimulator of interferon genes) but not the IMD (immune deficiency) signaling pathway. Collectively, our studies uncover a novel biological function of aspirin in modulating Drosophila antiviral immunity and provide theoretical bases for exploring new antiviral treatments in clinical trials.

Abstract Background Porcine epidemic diarrhea virus (PEDV) is a causative agent of serious viral enteric disease in suckling pigs. Such diseases cause considerable economic losses in the global swine industry. Enhancing our knowledge of PEDV-induced transcriptomic responses in host cells is imperative to understanding the molecular mechanisms involved in the immune response. Here, we analyzed the transcriptomic profile of intestinal porcine epithelial cell line J2 (IPEC-J2) after infection with a classical strain of PEDV to explore the host response. Results In total, 854 genes were significantly differentially expressed after PEDV infection, including 716 upregulated and 138 downregulated genes. Functional annotation analysis revealed that the differentially expressed genes were mainly enriched in the influenza A, TNF signaling, inflammatory response, cytokine receptor interaction, and other immune-related pathways. Next, the putative promoter regions of the 854 differentially expressed genes were examined for the presence of transcription factor binding sites using the MEME tool. As a result, 504 sequences (59.02%) were identified as possessing at least one binding site of signal transducer and activator of transcription (STAT), and five STAT transcription factors were significantly induced by PEDV infection. Furthermore, we revealed the regulatory network induced by STAT members in the process of PEDV infection. Conclusion Our transcriptomic analysis described the host genetic response to PEDV infection in detail in IPEC-J2 cells, and suggested that STAT transcription factors may serve as key regulators in the response to PEDV infection. These results further our understanding of the pathogenesis of PEDV.

With the development of high-throughput genotyping techniques, selection signatures in the genome of domestic pigs have been extensively interrogated in the last decade. The Duroc, a major commercial pig breed famous for its fast growth rate and high lean ratio, has not been extensively studied focusing on footprints of intensively artificial selection in their genomes by a lot of re-sequencing data. The goal of this study was to investigate genomic regions under artificial selection and their contribution to the unique phenotypic traits of the Duroc using whole-genome resequencing data from 97 pigs. Three complementary methods (di, CLR, and iHH12) were implemented for selection signature detection. In Total, 464 significant candidate regions were identified, which covered 46.4 Mb of the pig genome. Within the identified regions, 709 genes were annotated, including 600 candidate protein-coding genes (486 functionally annotated genes) and 109 lncRNA genes. Genes undergoing selective pressure were significantly enriched in the insulin resistance signaling pathway, which may partly explain the difference between the Duroc and other breeds in terms of growth rate. The selection signatures identified in the Duroc population demonstrated positive pressures on a set of important genes with potential functions that are involved in many biological processes. The results provide new insights into the genetic mechanisms of fast growth rate and high lean mass, and further facilitate follow-up studies on functional genes that contribute to the Duroc’s excellent phenotypic traits.

Abstract Background Reproductive performance of livestock is an economically important aspect of global food production. The Chinese Meishan pig is a prolific breed, with an average of three to five more piglets per litter than European breeds; however, the genetic basis for this difference is not well understood. Results In this study, we investigated copy number variations (CNVs) of 32 Meishan pigs and 29 Duroc pigs by next-generation sequencing. A genome-wide analysis of 61 pigs revealed 12,668 copy number variable regions (CNVRs) that were further divided into three categories based on copy number (CN) of the whole population, i.e., gain (n = 7,638), and loss (n = 5,030) CNVRs. We then compared Meishan and Duroc pigs and identified 17.17 Mb of 6,387 CNVRs that only existing in Meishan pigs CNVRs that overlapped the reproduction-related gene encoding the aryl hydrocarbon receptor (AHR) gene. We found that normal AHR CN was more frequent than CN loss in four different pig breeds. An association analysis showed that AHR CN had a positive effect on litter size (P

N-hexane, a common industrial organic solvent, causes multiple organ damage owing to its metabolite, 2,5-hexanedione (2,5-HD). To identify and evaluate the effects of 2,5-HD on sows’ reproductive performance, we used porcine ovarian granulosa cells (pGCs) as a vehicle and carried out cell morphology and transcriptome analyses. 2,5-HD has the potential to inhibit the proliferation of pGCs and induce morphological changes and apoptosis depending on the dose. RNA-seq analyses identified 4817 differentially expressed genes (DEGs), with 2394 down-regulated and 2423 up-regulated following 2,5-HD exposure treatment. The DEG, cyclin-dependent kinase inhibitor 1A (CDKN1A), according to the Kyoto Encyclopedia of Genes and Genomes enrichment analysis, was significantly enriched in the p53 signaling pathway. Thus, we evaluated its function in pGC apoptosis in vitro. Then, we knocked down the CDKN1A gene in the pGCs to identify its effects on pGCs. Its knockdown decreased pGC apoptosis, with significantly fewer cells in the G1 phase (p < 0.05) and very significantly more cells in the S phase (p < 0.01). Herein, we revealed novel candidate genes that influence pGCs apoptosis and cell cycle and provided new insights into the role of CDKN1A in pGCs during apoptosis and cell cycle arrest.

Structural variants (SVs) represent essential forms of genetic variation, and they are associated with various phenotypic traits in a wide range of important livestock species. However, the distribution of SVs in the pig genome has not been fully characterized, and the function of SVs in the economic traits of pig has rarely been studied, especially for most domestic pig breeds. Meishan pig is one of the most famous Chinese domestic pig breeds, with excellent reproductive performance. Here, to explore the genome characters of Meishan pig, we construct an SV map of porcine using whole-genome sequencing data and report 33,698 SVs in 305 individuals of 55 globally distributed pig breeds. We perform selective signature analysis using these SVs, and a number of candidate variants are successfully identified. Especially for the Meishan pig, 64 novel significant selection regions are detected in its genome. A 140-bp deletion in the Indoleamine 2,3-Dioxygenase 2 (IDO2) gene, is shown to be associated with reproduction traits in Meishan pig. In addition, we detect two duplications only existing in Meishan pig. Moreover, the two duplications are separately located in cytochrome P450 family 2 subfamily J member 2 (CYP2J2) gene and phospholipase A2 group IVA (PLA2G4A) gene, which are related to the reproduction trait. Our study provides new insights into the role of selection in SVs' evolution and how SVs contribute to phenotypic variation in pigs.

Abstract Background Mutations in mitochondrial tRNA genes have been widely reported association with human reproductions. It is also important to explore the effect on the number of piglets born alive (NBA). Here, 1017 sows were used to investigate the association between polymorphisms in mitochondrial tRNA genes and NBA. Results In total, 16 mutations were found in mitochondrial tRNA genes, of which 13 mutations were significantly associated with NBA (P

Abstract Background Pseudo-phenotypes, such as 305-day yields, estimated breeding values or deregressed proofs, are usually used as response variables for genome-wide association studies (GWAS) of milk production traits in dairy cattle. Computational inefficiency challenges the direct use of test-day records for longitudinal GWAS with large datasets. Results We propose a rapid longitudinal GWAS method that is based on a random regression model. Our method uses Eigen decomposition of the phenotypic covariance matrix to rotate the data, thereby transforming the complex mixed linear model into weighted least squares analysis. We performed a simulation study that showed that our method can control type I errors well and has higher power than a longitudinal GWAS method that does not include time-varied additive genetic effects. We also applied our method to the analysis of milk production traits in the first three parities of 6711 Chinese Holstein cows. The analysis for each trait was completed within 1 day with known variances. In total, we located 84 significant single nucleotide polymorphisms (SNPs) of which 65 were within previously reported quantitative trait loci (QTL) regions. Conclusions Our rapid method can control type I errors in the analysis of longitudinal data and can be applied to other longitudinal traits. We detected QTL that were for the most part similar to those reported in a previous study in Chinese Holstein. Moreover, six additional SNPs for fat percentage and 13 SNPs for protein percentage were identified by our method. These additional 19 SNPs could be new candidate quantitative trait nucleotides for milk production traits in Chinese Holstein.

Small RNA duodenal libraries were constructed for Escherichia coli F18-sensitive and -resistant weaned piglets in full-sib pair groups and sequenced using Illumina Solexa high-throughput sequencing technology. The identification of differentially expressed miRNAs provides the basis for improved database information on pig miRNAs, understanding the genetic basics of differences in resistance to E. coli F18 between local Chinese and exotic pig breeds, and finding new resistance markers for E. coli F18 infection. The duodenum of all individuals contained more than 90% of known swine miRNAs. A total of 58 differentially expressing miRNAs were identified, of which 46 were increased and 12 were decreased in E. coli F18-sensitive pigs. Of miRNAs with increased expression, ssc-miR-143 was most highly expressed, followed by ssc-let-7f, ssc-miR-192, and ssc-miR-21. We identified a total of 2036 intersection target genes by comparing TargetScan data and previous gene expression profile results. Gene ontology and pathway analysis of intersection genes showed that differentially expressed miRNAs were mainly involved in the immune response and transcriptional regulation. Combining information on differential miRNA expression and their regulatory relationships with transcription factors, identified 12 candidate miRNA disease markers, including 11 miRNAs with increased expression, ssc-miR-143, ssc-let-7f, ssc-miR-30e, ssc-miR-148a, ssc-miR-148b, ssc-miR-181a, ssc-miR-192, ssc-miR-27b, ssc-miR-15b, ssc-miR-21, and ssc-miR-215, and one with decreased expression, ssc-miR-152. Quantitative real-time PCR analysis of candidate miRNA expression in a larger cohort of E coli F18-sensitive and -resistant animals confirmed the high-throughput sequencing results.