Pathogen stimulations and immune cells synergistically affect the gene expression profile characteristics of porcine peripheral blood mononuclear cells.

BACKGROUND: Pigs serve as a crucial source of protein in the human diet and play a fundamental role in ensuring food security. However, infectious diseases caused by bacteria or viruses are a major threat to effective global pig farming, jeopardizing human health. Peripheral blood mononuclear cells (PBMCs) are a mixture of immune cells that play crucial roles in immunity and disease resistance in pigs. Previous studies on the gene expression regulation patterns of PBMCs have concentrated on a single immune stimulus or immune cell subpopulation, which has limited our comprehensive understanding of the mechanisms of the pig immune response. RESULTS: Here, we integrated and re-analyzed RNA-seq data published online for porcine PBMC stimulated by lipopolysaccharide (LPS), polyinosinic acid (PolyI:C), and various unknown microorganisms (EM). The results revealed that gene expression and its functional characterization are highly specific to the pathogen, identifying 603, 254, and 882 pathogen-specific genes and 38 shared genes, respectively. Notably, LPS and PolyI:C stimulation directly triggered inflammatory and immune-response pathways, while exposure to mixed microbes (EM) enhanced metabolic processes. These pathogen-specific genes were enriched in immune trait-associated quantitative trait loci (QTL) and eGenes in porcine immune tissues and were implicated in specific cell types. Furthermore, we discussed the roles of eQTLs rs3473322705 and rs1109431654 in regulating pathogen- and cell-specific genes CD300A and CD93, using cellular experiments. Additionally, by integrating genome-wide association studies datasets from 33 complex traits and diseases in humans, we found that pathogen-specific genes were significantly enriched for immune traits and metabolic diseases. CONCLUSIONS: We systematically analyzed the gene expression profiles of the three stimulations and demonstrated pathogen-specific and cell-specific gene regulation across different stimulations in porcine PBMCs. These findings enhance our understanding of shared and distinct regulatory mechanisms of genetic variants in pig immune traits.

Comprehensive analysis of circular RNAs in porcine small intestine epithelial cells associated with susceptibility to Escherichia coli F4ac diarrhea.

BACKGROUND: Diarrhea is one of the most common diseases in pig industry, which seriously threatens the health of piglets and causes huge economic losses. Enterotoxigenic Escherichia coli (ETEC) F4 is regarded as the most important cause of diarrhea in piglets. Some pigs are naturally resistant to those diarrheas caused by ETEC-F4, because they have no F4 receptors (F4R) on their small intestine epithelial cells that allow F4 fimbriae adhesion. Circular RNA (circRNA) has been shown to play an important regulatory role in the pathogenesis of disease. We hypothesized that circRNAs may also regulate the adhesion of piglet small intestinal epithelial cells to ETEC F4 fimbriae. However, the circRNA expression profiles of piglets with different Enterotoxigenic Escherichia coli F4 fimbriae (ETEC-F4ac) adhesion phenotypes are still unclear, and the intermediate regulatory mechanisms need to be explored. Hence, the present study assessed the circRNA expression profiling in small intestine epithelial cells of eight male piglets with different ETEC-F4 adhesion phenotypes and ITGB5 genotypes to unravel their regulatory function in susceptibility to ETEC-F4ac diarrhea. Piglets were divided into two groups: non-adhesive group (n = 4) with CC genotype and adhesive group (n = 4) with TT genotype. RESULTS: The RNA-seq data analysis identified 13,199 circRNAs from eight samples, most of which were exon-derived. In the small intestine epithelial cells, 305 were differentially expressed (DE) circRNAs between the adhesive and non-adhesive groups; of which 46 circRNAs were upregulated, and 259 were downregulated. Gene ontology and KEGG enrichment analysis revealed that most significantly enriched DE circRNAs' host genes were linked to cytoskeletal components, protein phosphorylation, cell adhesion, ion transport and pathways (such as adherens junction, gap junction) associated with ETEC diarrhea. The circRNA-miRNA-mRNA interaction network was also constructed to elucidate their underlying regulatory relationships. Our results identified several candidate circRNAs that affects susceptibility to ETEC diarrhea. Among them, circ-SORBS1 can adsorb ssc-miR-345-3p to regulate the expression of its host gene SORBS1, thus improving cell adhesion. CONCLUSION: Our results provided insights into the regulation function of circRNAs in susceptibility to ETEC diarrhea of piglets, and enhanced our understanding of the role of circRNAs in regulating ETEC diarrhea, and reveal the great potential of circRNA as a diagnostic marker for susceptibility of ETEC diarrhea in piglets.

Application and prospect of gene chip in genetic breeding of livestock and poultry.

Gene chip is a high-throughput technique for detecting specific DNA sequences by DNA or DNA-RNA complementary hybridization, among which SNP genotyping chips have been widely employed in the animal genetics and breeding, and have made great achievements in cattle (Bos taurus), pigs (Sus scrofa), sheep (Caprinae), chickens (Gallus gallus) and other livestock. However, genomic selection applied in production merely uses genomic information and cannot fully explain the molecular mechanism of complex traits genetics, which limits the accuracy of genomic selection. With the continuous progresses in epigenetic research, the development of commercial methylation chips and the application of the epigenome-wide association study (EWAS), DNA methylation has been extensively used to draw the causal connections between genetics and phenotypes. In the future, it is hopefully expected to develop methylation chips customized for livestock and poultry and explore methylation sites significantly related to economic traits of livestock and poultry through EWAS thereby extending the understanding of causal variation of complex traits. Combining methylation chips and SNP chips, we can capture the epigenomic and genomic information of livestock and poultry, interpret genetic variation more precisely, improve the accuracy of genome selection, and promote the fine evolution of molecular genetic breeding of livestock and poultry. In this review, we summarize the application of SNP chips and depict the prospects of the application of methylation chips in livestock and poultry. This review will provide valuable insights for further application of gene chips in farm animal breeding.

Feed tossing behaviour of Holstein cows: evaluation of physiological stress state and rumen fermentation function.

BACKGROUND: Abnormal or stereotyped behaviours in dairy cows are common in large-scale indoor farms and are usually accompanied by high physiological stress levels. Feed tossing is an abnormal behaviour commonly seen in cows while being fed, making farm management difficult. However, the reasons behind this behaviour have not been sufficiently reported. The objective of this study was to explore the changes in rumen fermentation, serum indicators, inflammatory conditions and the performance of cows with feed tossing behaviour. Holstein cows with similar lactation stages in the same barn were subjected to behaviour observations two times per day for 21 consecutive days. Ten cows with feed tossing behaviour (FT) and ten cows without abnormal behaviours (CON) were selected for further sampling. Plasma samples, rumen fluid, milk yield data of cows, and an indoor environment temperature-humidity index (THI) were collected. RESULTS: There was no significant difference in average daily milk yield during the observation period between feed-tossing cows (n = 68) and the other cows (n = 112). The number of cows showing FT behaviour had a moderately strong negative linear correlation with the THI of the environment. Compared to the CON cows, the FT cows had higher cortisol, norepinephrine and urea nitrogen levels in plasma, as well as higher plasma levels of inflammatory indicators, including total protein, lactate dehydrogenase, albumin, aspartate aminotransferase levels, and the ratio of aspartate aminotransferase to alanine aminotransferase. The FT cows had no significant variations from the CON cows regarding their rumen fermentation indicators, such as pH, ammonia nitrogen, and volatile fatty acids. In addition, 16S rRNA analysis revealed that there might be no clear association between the diversity and abundance of rumen bacteria and feed tossing behaviour. CONCLUSIONS: Our findings suggested that cows might have suffered from high levels of physiological stress and immune state for a long period when they exhibited FT behaviour. The environmental THI could affect the FT behaviour of cows; as the THI increases, the willingness of cows to throw decreases. This work provided the first evidence that feed tossing might be a response associated with high levels of physiological stress and immune. It also explored our insights into a commonly observed behavioural response to cow welfare traits.

[Analysis of endogenous plasmids in Lacticaseibacillus paracasei ZY-1 and development of expression vectors].

The construction of efficient and stable Lactobacillus expression vector is critical for strain improvement and development of customized strains. In this study, four endogenous plasmids were isolated from Lacticaseibacillus paracasei ZY-1 and subjected to functional analysis. The Escherichia coli-Lactobacillus shuttle vectors pLPZ3N and pLPZ4N were constructed by combining the replicon rep from pLPZ3 or pLPZ4, the chloramphenicol acetyltransferase gene cat from pNZ5319 and the replicon ori from pUC19. Moreover, the expression vectors pLPZ3E and pLPZ4E with the promoter Pldh3 of lactic acid dehydrogenase and the mCherry red fluorescent protein as a reporter gene were obtained. The size of pLPZ3 and pLPZ4 were 6 289 bp and 5 087 bp, respectively, and its GC content, 40.94% and 39.51%, were similar. Both shuttle vectors were successfully transformed into Lacticaseibacillus, and the transformation efficiency of pLPZ4N (5.23×102-8.93×102 CFU/µg) was slightly higher than that of pLPZ3N. Furthermore, the mCherry fluorescent protein was successfully expressed after transforming the expression plasmids pLPZ3E and pLPZ4E into L. paracasei S-NB. The ß-galactosidase activity of the recombinant strain obtained from the plasmid pLPZ4E-lacG constructed with Pldh3 as promoter was higher than that of the wild-type strain. The construction of shuttle vectors and expression vectors provide novel molecular tools for the genetic engineering of Lacticaseibacillus strains.

Physiological Indicators and Production Performance of Dairy Cows With Tongue Rolling Stereotyped Behavior.

Non-nutritive oral behaviors, especially tongue rolling, are prevalent in the stabled cow population. These behaviors mean that the environment or management process might not suit the cows, suggesting low welfare. However, few researches have reported the physiological indicators or production performance of dairy cows with the stereotyped behavior. This study aimed to determine physical conditions, daily activity, rumen fermentation, and milk production of cows with tongue-rolling behavior. Three hundred and fifty nine Holstein cows in the same barn and lactation stage were subjected to scan sampling behavior observations 126 times for 7 days. Ten cows with high-frequency tongue-rolling behavior (TON) and 10 cows without abnormal oral behavior (CON) were selected for further study. Serum sample, ruminal fluid, milk sample, and behavior record video of TON and CON cows were collected. TON cows had more drinking behavior and more stable lying behavior than the CON cows during the daytime. The body condition score of the TON cows decreased, while the milk yield, yield of milk fat, protein, and lactose in the study period increased. The TON cows had lower ruminal fluid pH, acetate/propionate ratio, and total volatile acid. The bacterial diversity in the ruminal fluid was not different between the two groups. Compared to CON cows, the TON cows had a higher level of serum stress indicators, such as cortisol, thyroid hormone, and norepinephrine, which positively correlated to the frequency of tongue-rolling behavior. Meanwhile, the TON cows had a higher level of lactate dehydrogenase, serum glucose, total triglyceride, total cholesterol, and Interleukin 6. Overall, it means they suffer from higher levels of stress and have higher energy metabolism for a long time when cows show tongue-rolling behavior. TON cows had suffered a higher stress level and had higher energy metabolic status for a long time. The TON cows might have better heat tolerance to the thermal environment by more lying and drinking time. Our data revealed the changes in milk production, physiological stress indicators of dairy cows with high-frequency tongue rolling behavior, which will provide essential knowledge for the in-depth understanding of tongue rolling behavior in dairy cows.

Metabolic Profiling of Plasma in Different Calving Body Condition Score Cows Using an Untargeted Liquid Chromatography-Mass Spectrometry Metabolomics Approach.

This study was undertaken to identify metabolite differences in plasma of dairy cows with a normal or high calving body condition score (CBCS), using untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomics. Sixteen multiparous dairy cows were assigned to one of two groups based on CBCS (0 to 5 scale): Normal group (NBCS, 3.25 ≤ BCS ≤ 3.5, n = 8), and high BCS group (HBCS, BCS ≥ 4, n = 8). Plasma samples were collected for metabolomics analysis and evaluation of biomarkers of lipid metabolism (nonesterified fatty acid (NEFA) and ß-hydroxybutyrate (BHB)), and cytokines (leptin, adiponectin, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6)). A total of 23 differential metabolites were identified, and functional analyses were performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Among these metabolites, the concentrations of six lysophosphatidylcholines and one phosphatidylethanolamine, were lower in the HBCS group than in the NBCS group (p < 0.01). Furthermore, these metabolites were involved in these four pathways, among others: glycerophospholipid metabolism, retrograde endocannabinoid signaling, autophagy, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis (p < 0.05). In addition, plasma concentrations of leptin (p = 0.06) and TNF-α (p = 0.08) tended to be greater while adiponectin (p = 0.09) lower in HBCS cows than in NBCS cows. The concentrations of NEFA, BHB, or IL-6 did not differ between NBCS and HBCS groups. More importantly, based on the results of the Spearman's correlation analysis, the seven important metabolites were negatively correlated with indices of lipid metabolisms, proinflammatory cytokines, and leptin, but positively correlated with adiponectin. These results demonstrate that CBCS has a measurable impact on the plasma metabolic profile, even when NEFA and BHB are not different. In addition, the identified differential metabolites were significantly correlated to lipid metabolism and inflammation in the over-conditioned fresh cows, which are expected to render a metabolic basis for the diseases associated with over-conditioned dry cows.