Rotavirus infection inhibits SLA-I expression on the cell surface by degrading ß2 M via ERAD-proteasome pathway.

Group A Rotavirus (RVA) is a major cause of diarrhea in infants and piglets. ß2-microglobulin (ß2 M), encoded by the B2M gene, serves as a crucial subunit of the major histocompatibility complex class I (MHC-I) molecules. ß2 M is indispensable for the transport of MHC-I to the cell membrane. MHC-I, also known as swine leukocyte antigen class I (SLA-I) in pigs, presents viral antigens to the cell surface. In this study, RVA infection down-regulated ß2 M expression in both porcine intestinal epithelial cells-J2 (IPEC-J2) and MA-104 cells. RVA infection did not down-regulate the mRNA level of the B2M gene, indicating that the down-regulation of ß2 M occurred on the protein level. Mechanismly, RVA infection triggered ß2 M aggregation in the endoplasmic reticulum (ER) and enhanced the Lys48 (K48)-linked ubiquitination of ß2 M, leading to the degradation of ß2 M through ERAD-proteasome pathway. Furthermore, we found that RVA infection significantly impeded the level of SLA-I on the surface, and the overexpression of ß2 M could recover its expression. In this study, our study demonstrated that RVA infection degrades ß2 M via ERAD-proteasome pathway, consequently hampering SLA-I expression on the cell surface. This study would enhance the understanding of the mechanism of how RVA infection induces immune escape.

Enteric coronavirus PDCoV evokes a non-Warburg effect by hijacking pyruvic acid as a metabolic hub.

The Warburg effect, also referred as aerobic glycolysis, is a common metabolic program during viral infection. Through targeted metabolomics combined with biochemical experiments and various cell models, we investigated the central carbon metabolism (CCM) profiles of cells infected with porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus with zoonotic potential. We found that PDCoV infection required glycolysis but decreased glycolytic flux, exhibiting a non-Warburg effect characterized by pyruvic acid accumulation. Mechanistically, PDCoV enhanced pyruvate kinase activity to promote pyruvic acid anabolism, a process that generates pyruvic acid with concomitant ATP production. PDCoV also hijacked pyruvic acid catabolism to increase biosynthesis of non-essential amino acids (NEAAs), suggesting that pyruvic acid is an essential hub for PDCoV to scavenge host energy and metabolites. Furthermore, PDCoV facilitated glutaminolysis to promote the synthesis of NEAA and pyrimidines for optimal proliferation. Our work supports a novel CCM model after viral infection and provides potential anti-PDCoV drug targets.

Large animal models for Huntington's disease research.

Huntington's disease (HD) is a hereditary neurodegenerative disorder for which there is currently no effective treatment available. Consequently, the development of appropriate disease models is critical to thoroughly investigate disease progression. The genetic basis of HD involves the abnormal expansion of CAG repeats in the huntingtin ( HTT) gene, leading to the expansion of a polyglutamine repeat in the HTT protein. Mutant HTT carrying the expanded polyglutamine repeat undergoes misfolding and forms aggregates in the brain, which precipitate selective neuronal loss in specific brain regions. Animal models play an important role in elucidating the pathogenesis of neurodegenerative disorders such as HD and in identifying potential therapeutic targets. Due to the marked species differences between rodents and larger animals, substantial efforts have been directed toward establishing large animal models for HD research. These models are pivotal for advancing the discovery of novel therapeutic targets, enhancing effective drug delivery methods, and improving treatment outcomes. We have explored the advantages of utilizing large animal models, particularly pigs, in previous reviews. Since then, however, significant progress has been made in developing more sophisticated animal models that faithfully replicate the typical pathology of HD. In the current review, we provide a comprehensive overview of large animal models of HD, incorporating recent findings regarding the establishment of HD knock-in (KI) pigs and their genetic therapy. We also explore the utilization of large animal models in HD research, with a focus on sheep, non-human primates (NHPs), and pigs. Our objective is to provide valuable insights into the application of these large animal models for the investigation and treatment of neurodegenerative disorders.

RBM14 inhibits the replication of porcine epidemic diarrhea virus by recruiting p62 to degrade nucleocapsid protein through the activation of autophagy and interferon pathway.

Porcine epidemic diarrhea virus (PEDV) results in PED, which is an infectious intestinal disease with the representative features of diarrhea, vomiting, and dehydration. PEDV infects neonatal piglets, causing high mortality rates. Therefore, elucidating the interaction between the virus and host in preventing and controlling PEDV infection is of immense significance. We found a new antiviral function of the host protein, RNA-binding motif protein 14 (RBM14), which can inhibit PEDV replication via the activation of autophagy and interferon (IFN) signal pathways. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV nucleocapsid (N) protein through the RBM14-p62-autophagosome pathway. Furthermore, RBM14 can also improve the antiviral ability of the hosts through interacting with mitochondrial antiviral signaling protein to induce IFN expression. These results highlight the novel mechanism underlying RBM14-induced viral restriction. This mechanism leads to the degradation of viral N protein via the autophagy pathway and upregulates IFN for inhibiting PEDV replication; thus, offering new ways for preventing and controlling PED.IMPORTANCEPorcine epidemic diarrhea virus (PEDV) is a vital reason for diarrhea in neonatal piglets, which causes high morbidity and mortality rates. There is currently no effective vaccine or drug to treat and prevent infection with the PEDV. During virus infection, the host inhibits virus replication through various antiviral factors, and at the same time, the virus antagonizes the host's antiviral reaction through its own encoded protein, thus completing the process of virus replication. Our study has revealed that the expression of RNA-binding motif protein 14 (RBM14) was downregulated in PEDV infection. We found that RBM14 can recruit cargo receptor p62 to degrade PEDV N protein via the RBM14-p62-autophagosome pathway and interacted with mitochondrial antiviral signaling protein and TRAF3 to activate the interferon signal pathway, resulting in the inhibition of PEDV replication.

SADS-CoV nsp1 inhibits the STAT1 phosphorylation by promoting K11/K48-linked polyubiquitination of JAK1 and blocks the STAT1 acetylation by degrading CBP.

The newly discovered zoonotic coronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) causes acute diarrhea, vomiting, dehydration, and high mortality rates in newborn piglets. Although SADS-CoV uses different strategies to evade the host's innate immune system, the specific mechanism(s) by which it blocks the interferon (IFN) response remains unidentified. In this study, the potential of SADS-CoV nonstructural proteins (nsp) to inhibit the IFN response was detected. The results determined that nsp1 was a potent antagonist of IFN response. SADS-CoV nsp1 efficiently inhibited signal transducer and activator of transcription 1 (STAT1) phosphorylation by inducing Janus kinase 1 (JAK1) degradation. Subsequent research revealed that nsp1 induced JAK1 polyubiquitination through K11 and K48 linkages, leading to JAK1 degradation via the ubiquitin-proteasome pathway. Furthermore, SADS-CoV nsp1 induced CREB-binding protein degradation to inhibit IFN-stimulated gene production and STAT1 acetylation, thereby inhibiting STAT1 dephosphorylation and blocking STAT1 transport out of the nucleus to receive antiviral signaling. In summary, the results revealed the novel mechanisms by which SADS-CoV nsp1 blocks the JAK-STAT signaling pathway via the ubiquitin-proteasome pathway. This study yielded valuable findings on the specific mechanism of coronavirus nsp1 in inhibiting the JAK-STAT signaling pathway and the strategies of SADS-CoV in evading the host's innate immune system.

Potassium diformate alleviated inflammation of IPEC-J2 cells infected with EHEC.

Potassium diformate (KDF) is a kind of formate, which possesses the advantages of antimicrobial activity, growth promotion and preventing diarrhea in weaned piglets. However, the researches of KDF in animal production mostly focused on apparent indexes such as growth performance and the mechanisms of KDF on intestinal health have not been reported. Thus, porcine small intestinal epithelial cells (IPEC-J2) infected with Enterohemorrhagic Escherichia coli (EHEC) was used to investigate the role of KDF on alleviating intestinal inflammation in this study. The 0.125 mg/mL KDF treated IPEC-J2 cells for 6 h and IPEC-J2 cells challenged with 5 × 107 CFU/mL EHEC for 4 h were confirmed as the optimum concentration and time for the following experiment. The subsequent experiment was divided into four groups: control group (CON), EHEC group, KDF group, KDF+EHEC group. The results showed that KDF increased the cell viability and the gene expression levels of SGLT3 and TGF-ß, while decreased the content of IL-1ß compared with the CON group. The cell viability and the gene expressions of SGLT1, SGLT3, GLUT2, Claudin-1, Occludin and TGF-ß, and the protein expression of ZO-1 in EHEC group were lower than those in CON group, whereas the gene expressions of IL-1ß, TNF, IL-8 and TLR4, and the level of phosphorylation NF-кB protein were increased. Pretreatment with KDF reduced the content of IgM and IL-1ß, the gene expressions of IL-1ß, TNF, IL-8 and TLR4 and the level of phosphorylation NF-кB protein, and increased the gene expression of TGF-ß and the protein expression of Occludin in IPEC-J2 cells infected EHEC. In conclusion, 0.125 mg/mL KDF on IPEC-J2 cells for 6 h had the beneficial effects on ameliorating the intestinal inflammation because of reduced pro-inflammatory cytokines and enhanced anti-inflammatory cytokines through regulating NF-кB signaling pathway under the EHEC challenge.

S100A12 protein as a porcine health status biomarker when quantified in saliva samples.

The S100A12 protein was validated as a biomarker of health status in porcine saliva samples using a semi-quantitative approach based on Western blotting in four healthy and sixteen diseased animals, and in four animals with severe respiratory disease during three days of antibiotic therapy. Afterwards, a non-competitive sandwich immunoassay was then developed, validated, and used to quantify S100A12 in clinical porcine samples, using 14 healthy and 25 diseased pigs. Finally, the S100A12 concentrations in the saliva of ten pigs with respiratory disease were monitored during antibiotic therapy. Diseased animals showed higher concentrations of S100A12 than healthy animals, and the high concentrations of S100A12 in pigs with respiratory distress were reduced after antimicrobial therapy. The assay developed showed good precision and accuracy, as well as a low limit of detection of 3.19 ng/mL. It was possible to store saliva samples at -20 °C, or even at 4 °C, for two weeks before analysis without losing the validity of the results. The concentrations of S100A12 observed in serum and saliva samples showed a moderately positive association with a correlation coefficient of 0.48. The concentrations of the new validated biomarker S100A12 are highly associated with the novel salivary biomarker of inflammation, adenosine deaminase, and moderately to highly associated with the total oxidant status. The results reported in this study provide a new way of evaluating inflammatory diseases in pigs using saliva samples, which should be further explored for disease prevention and monitoring in the field.

Implications for miR-339-5p regulation of trophoblast proliferation and migration in placentas associated with porcine intrauterine growth retardation using integrated transcriptome sequencing analysis.

Placental dysfunction is considered as one of the main etiologies of fetal intrauterine growth retardation (IUGR). MicroRNAs (miRNAs) have been demonstrated to be a vital epigenetic modification involved in regulating the placental function and pregnancy outcomes in mammals. However, the mechanisms underlying placenta-specific miRNAs involved in the occurrence and development of pig IUGR remain unclear. In this work, we compared the placental morphologies of piglets with IUGR and normal birth weight (NBW) by using histomorphological analysis and performed a miRNA-mRNA integrative analysis of the gene expression profiles of IUGR and NBW placentas through RNA sequencing. We also investigated the role of differentially expressed ssc-miR-339-5p/GRIK3 through an in vitro experiment on porcine trophoblast cells (PTr2). IUGR piglets had significantly lower birth weight, placental weight, placental efficiency, and placental villus and capillary densities compared with the NBW piglets (P < 0.05). A total of 81 differentially expressed miRNAs and 726 differentially expressed genes in the placentas were screened out between the IUGR and NBW groups. The miRNA-mRNA interaction networks revealed the key core miRNA (ssc-miR-339-5p) and its corresponding target genes. Subsequently, we found that upregulation of ssc-miR-339-5p significantly inhibited the migration and proliferation of PTr2 cells (P < 0.05). The dual-luciferase reporter system showed that GRIK3 was the target gene of ssc-miR-339-5p, and the transcription level of GRIK3 may be negatively regulated by ssc-miR-339-5p. Additionally, overexpression of ssc-miR-339-5p significantly increased (P < 0.05) the mRNA expression levels of genes involved in the cytokine-cytokine receptor interaction pathway. These results indicate that ssc-miR-339-5p may affect the migration and proliferation of trophoblast cells by regulating the expression of GRIK3 and altering the placental inflammatory response, resulting in a suboptimal morphology and function of the placenta and the development of pig IUGR.

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3.

Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

Infection of PRRSV inhibits CSFV C-strain replication by inducing macrophages polarization to M1.

It is a common sense that porcine reproductive and respiratory syndrome virus (PRRSV) infection could cause immune failure of classical swine fever (CSF) vaccine, and porcine alveolar macrophages (PAMs) are the target cells of both. To elucidate the role of macrophage polarization in PRRSV infection induced CSF vaccine failure, an immortal porcine alveolar macrophage line PAM39 cell line was used to investigate the effect of PRRSV or/and CSFV C-strain (CSFV-C) infection on macrophage polarization in vitro. Interestingly, PRRSV single infection or PRRSV co-infection with CSFV-C promoted PAM39 cells to M1, while CSFV-C single infection induced PAM39 cells to M2. After the construction of M1 and M2 PAM39 cells polarization models, M1 polarized PAM39 cells were found to inhibit the replication of CSFV-C, and Chinese medicine such as matrine, ginsenosides and astragalus polysaccharides could alleviate the polarization of PAM39 cells and the replication of CSFV-C. Furthermore, interferon (IFN)-γ and lipopolysaccharide (LPS) co-stimulation induced NF-κB activation while matrine treatment blocked M1 polarization-induced NF-κB pathway activation. These findings provided a theoretical basis for designing a new strategy to improve the immune effect of CSFV-C based on porcine alveolar macrophage polarization subtypes.

O-acetyl-homoserine sulfhydrylase deficient Streptococcus suis serotype 2 strain SC19 becomes an avirulent strain and provides immune protection against homotype infection in mice.

O-acetyl-homoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis, which catalyzes the conversion of o-acetyl-homoserine (OAH) to homocysteine. In our previous study, we found that OAHS of Streptococcus suis serotype 2 (SS2) can interact with the porcine blood-brain barrier (BBB) model, but whether OAHS regulates the penetration of BBB during SS2 infection is still unclear. To explore the role of OAHS in SS2 infection, OAHS-deficient SS2 mutant strain (SC19-ΔOAHS) and gene complemental strain (SC19-cΔOAHS) were constructed. Compared to the parent strain, with the loss of oahs, the chain length of SC19-ΔOAHS was shortened, the virulence was significantly reduced, the survival rate of mice infected with SC19-ΔOAHS was obviously increased accompanied by the relieved clinical symptoms. And the survival ability of SC19-ΔOAHS in whole blood was also remarkably decreased. Interestingly, the adhesion of SC19-ΔOAHS to endothelial cells was markedly increased, but the deficiency of OAHS significantly inhibited the strain penetrating BBB both in vivo and in vitro. Most of these phenomena can be reversed by the complemental strain (SC19-cΔOAHS). Further study showed that the deficiency of OAHS severely reduced SC19-induced endothelial cell apoptosis, tight junctions (TJs) protein impairment and the expression of SS2 virulence factor Enolase (Eno), involved in the destruction of BBB. Additionally, SC19-ΔOAHS immunized mice were able to resist SC19 or JZLQ022 infection. In conclusion, we confirmed that OAHS promoted the pathogenicity by enhancing host's BBB permeability and immune escape, and SC19- ΔOAHS is a potential live vaccine.

Effects of dietary epidermal growth factor supplementation on liver antioxidant capacity of piglets with intrauterine growth retardation.

The present experiment was conducted to study the effects of dietary epidermal growth factor (EGF) supplementation on the liver antioxidant capacity of piglets with intrauterine growth retardation (IUGR). The present study consists of two experiments. In experiment 1, six normal-birth-weight (NBW) and six IUGR newborn piglets were slaughtered within 2 to 4 h after birth to compare the effects of IUGR on the liver antioxidant capacity of newborn piglets. The results showed that compared with NBW piglets, IUGR piglets had a lower birth weight and liver relative weight; IUGR piglets had a higher serum malondialdehyde (MDA) level, liver MDA level and hydrogen peroxide (H2O2) level, and had a lower liver total antioxidant capacity (T-AOC) level and glutathione peroxidase (GSH-Px) activity; IUGR trended to increase serum alanine aminotransferase activity, aspartate aminotransferase activity, and H2O2 level, and trended to decrease liver total superoxide dismutase activity. In experiment 2, six NBW piglets, and 12 IUGR piglets weaned at 21 d of age were randomly divided into the NC group (NBW piglets fed with basal diet); IC group (IUGR piglets fed with basal diet), and IE group (IUGR piglets fed with basal diet plus 2 mg/kg EGF), and feeding for 14 d. Organ index, serum parameters, liver antioxidant capacity, and liver antioxidant-related genes expression were measured. The results showed that compared to the IC group, dietary EGF supplementation (IE group) significantly reduced serum malondialdehyde level and H2O2 level, and liver protein carbonyl (PC) level and 8-hydroxydeoxyguanosine level of piglets with IUGR; dietary EGF supplementation (IE group) significantly increased serum T-AOC level, liver T-AOC level and GSH-Px activity; dietary supplemented with EGF (IE group) enhanced liver Nrf2, NQO1, HO1, and GPX1 mRNA expression compared to IC group. Pearson's correlation analysis further showed that EGF can alleviate liver oxidative injury caused by IUGR and improve the performance of IUGR piglets. In conclusion, EGF exhibited potent protective effects on IUGR-induced liver oxidative injury, by activating the Nrf2 signaling pathway to mediate the expression of downstream antioxidant enzymes and phase II detoxification enzymes (NQO1 and HO1), thereby alleviating liver oxidative damage and promoting the growth performance of IUGR piglets.

The liver is an important metabolic and secretory organ in vertebrates, which plays an important role in the overall health of animals. Studies have shown that intrauterine growth retardation (IUGR) can cause liver injury in piglets, which is unfavorable to the growth and development of piglets. Epidermal growth factor (EGF) has antioxidant properties, but its effect on liver oxidative damage caused by IUGR remains uncertain. In the present study, we chose newborn piglets with low birth weight as the IUGR models to investigate whether IUGR could cause oxidative damage in the liver. Then, the diet supplemented with EGF was fed to IUGR piglets to study the effects of EGF supplementation on the liver antioxidant function of IUGR-weaned piglets. Results showed that IUGR caused serious damage to the liver of piglets, while dietary EGF supplementation could reverse the oxidative injury induced by IUGR to some extent. Therefore, this study confirmed that EGF has positive effects on the liver health of piglets with IUGR.

Toxoplasma gondii infection regulates apoptosis of host cells via miR-185/ARAF axis.

BACKGROUND: Toxoplasmosis is a zoonosis with a worldwide presence that is caused by the intracellular parasite Toxoplasma gondii. Active regulation of apoptosis is an important immune mechanism by which host cells resist the growth of T. gondii or avoid excessive pathological damage induced by this parasite. Previous studies found that upregulated expression of microRNA-185 (miR-185) during T. gondii infection has a potential role in regulating the expression of the ARAF gene, which is reported to be associated with cell proliferation and apoptosis. METHODS: The expression levels of miR-185 and the ARAF gene were evaluated by qPCR and Western blot, respectively, in mice tissues, porcine kidney epithelial cells (PK-15) and porcine alveolar macrophages (3D4/21) following infection with the T. gondii ToxoDB#9 and RH strains. The dual luciferase reporter assay was then used to verify the relationship between miR-185 and ARAF targets in PK-15 cells. PK-15 and 3D4/21 cell lines with stable knockout of the ARAF gene were established by CRISPR, and then the apoptosis rates of the cells following T. gondii infection were detected using cell flow cytometry assays. Simultaneously, the activities of cleaved caspase-3, as a key apoptosis executive protein, were detected by Western blot to evaluate the apoptosis levels of cells. RESULTS: Infection with both the T. gondii ToxoDB#9 and RH strains induced an increased expression of miR-185 and a decreased expression of ARAF in mice tissues, PK-15 and 3D4/21 cells. MiR-185 mimic transfections showed a significantly negative correlation in expression levels between miR-185 and the ARAF gene. The dual luciferase reporter assay confirmed that ARAF was a target of miR-185. Functional investigation revealed that T. gondii infection induced the apoptosis of PK-15 and 3D4/21 cells, which could be inhibited by ARAF knockout or overexpression of miR-185. The expression levels of cleaved caspase-3 protein were significantly lower in cells with ARAF knockout than in normal cells, which were consistent with the results of the cell flow cytometry assays. CONCLUSIONS: Toxoplasma gondii infection could lead to the upregulation of miR-185 and the downregulation of ARAF, which was not related to the strain of T. gondii and the host cells. Toxoplasma gondii infection could regulate the apoptosis of host cells via the miR-185/ARAF axis, which represents an additional strategy used by T. gondii to counteract host-cell apoptosis in order to maintain survival and reproduce in the host cells.

Pork quality traits and associated muscle metabolic changes in pigs under chronic prenatal and postnatal heat stress.

Chronic heat stress (HS) is a major concern affecting pig growth performance and metabolism, with potential consequences on carcass and meat quality traits. The objective of this study was to assess the influence of prenatal (PE) and growing (GE) thermal environments, and their combination, on muscle metabolism, carcass characteristics, and pork quality. From 6 to 109 d of gestation, 12 sows (1 per block) were kept under thermoneutral (TN) conditions (cyclic 18 to 24 °C; PTN) and 12 sows under chronic HS (cyclic 28 to 34 °C; PHS). Two female offspring per sow were selected based on body weight at weaning, for a total of 48 female pigs (12 blocks of 2 sisters from each PE), and one sister was placed in each GE. Gilts were housed from 82 to 140 d of age under cyclic GTN (18 to 24 °C; n = 24) or GHS (28 to 34 °C; n = 24) environments. Data were analyzed using a mixed model including PE, GE, and PE × GE interaction as main effects, and sire, sow within PE, pen within PE × GE, and slaughter day (for plasma, muscle, and meat traits) as random effects. No significant PE × GE interaction was found on any trait under study (P ≥ 0.05). Prenatal HS did not affect growth performance and carcass traits (P ≥ 0.05). Compared with GTN, GHS pigs had lower average daily feed intake, average daily gain, and hot carcass weight (P < 0.01), but similar carcass lean meat content (P ≥ 0.05). Prenatal HS had scarce effects on pork quality, with only higher a* and C* values (P < 0.05) in the Gluteus superficialis. Growing HS led to a higher pH 24 h (P < 0.05) in the Longissimus thoracis et lumborum (LTL) and ham muscles, and higher meat quality index in the ham muscles. In contrast, quality traits of the Semispinalis capitis (SC) were not affected by either PE or GE (P > 0.05). Except a tendency for a higher citrate synthase activity in the SC (P = 0.065), PHS did not affect muscle metabolism. Growing HS induced muscle-specific metabolic responses, with reduced glycolytic potential (P < 0.01) and metabolic enzyme activities (P < 0.05) in the glycolytic LTL, but not in the oxidative SC (P > 0.05). Plasma glucose content at slaughter was lower in the GHS compared with GTN pigs (P = 0.002), indicating an altered energy metabolism in pigs under GHS. Altogether, growing HS altered growth without affecting carcass traits, but improved technological quality of loin and ham. Prenatal HS, alone or combined with GHS, had limited or even no effect on carcass and pork quality.

Pig thermal environment and especially chronic heat stress (HS) can affect animal growth and physiology, with potential impacts on carcass and pork quality. The effects of chronic HS during postnatal growth (GHS) on pork quality are controversial, but the effects of chronic HS during the pig prenatal period (PHS) or of combined PHS and GHS on carcass and meat traits are even less characterized. However, a better understanding of HS consequences on pork quality is of high economic importance for the pork sector, as the frequency and severity of heat waves are likely to increase with global warming. This research focused on the effects of PHS and GHS on pork carcass and meat quality from various muscles and primary cuts (loin, ham), and the underlying muscle biochemical properties. Prenatal HS did not affect growth and carcass traits. Compared with pigs grown in a thermoneutral environment, GHS pigs had reduced gain resulting from lower feed intake and had lighter carcasses, but similar carcass lean meat content. PHS had little effect on pork quality, whereas GHS induced specific metabolic effects in the loin and ham muscles, leading to higher meat pH and water-holding capacity and thereby improved pork technological quality.

N-linked glycoproteins and host proteases are involved in swine acute diarrhea syndrome coronavirus entry.

IMPORTANCE: Gaining insight into the cell-entry mechanisms of swine acute diarrhea syndrome coronavirus (SADS-CoV) is critical for investigating potential cross-species infections. Here, we demonstrated that pretreatment of host cells with tunicamycin decreased SADS-CoV attachment efficiency, indicating that N-linked glycosylation of host cells was involved in SADS-CoV entry. Common N-linked sugars Neu5Gc and Neu5Ac did not interact with the SADS-CoV S1 protein, suggesting that these molecules were not involved in SADS-CoV entry. Additionally, various host proteases participated in SADS-CoV entry into diverse cells with different efficiencies. Our findings suggested that SADS-CoV may exploit multiple pathways to enter cells, providing insights into intervention strategies targeting the cell entry of this virus.

Postmortem calpain changes in longissimus muscle from Lanyu native and Kaohsiung crossbred black pigs.

The purpose of this study was to compare postmortem calpain changes in porcine muscle between Lanyu native (n = 10) and Kaohsiung crossbred (50% Meishan and 50% Duroc, n = 10) black pigs. Longissimus dorsi (LM) muscles (4th to 10th ribs) from both sides of carcasses were hot-sectioned in 30 min postmortem, vacuum-packaged, and stored at 5°C. LM samples from the left side of carcasses were sampled at 0 (30 min postmortem), 3, 6, 12, 24, 48, and 72 h of storage. The 24 and 72 h samples of shear force measurement were taken from the LM at the 8th and 10th ribs of the right side of carcasses, respectively. Our results showed that the decreases in calpain-1 activity, desmin content, and shear force were slower (P < 0.05) in Lanyu native black pig than in Kaohsiung crossbred black pig samples. Therefore, postmortem proteolysis and tenderization were more limited in LM from Lanyu native pigs than from Kaohsiung crossbred pigs.

Melatonin supplementation promotes muscle fiber hypertrophy and regulates lipid metabolism of skeletal muscle in weaned piglets.

Melatonin has been reported to play crucial roles in regulating meat quality, improving reproductive properties, and maintaining intestinal health in animal production, but whether it regulates skeletal muscle development in weaned piglet is rarely studied. This study was conducted to investigate the effects of melatonin on growth performance, skeletal muscle development, and lipid metabolism in animals by intragastric administration of melatonin solution. Twelve 28-d-old DLY (Duroc × Landrace × Yorkshire) weaned piglets with similar body weight were randomly divided into two groups: control group and melatonin group. The results showed that melatonin supplementation for 23 d had no effect on growth performance, but significantly reduced serum glucose content (P < 0.05). Remarkably, melatonin increased longissimus dorsi muscle (LDM) weight, eye muscle area and decreased the liver weight in weaned piglets (P < 0.05). In addition, the cross-sectional area of muscle fibers was increased (P < 0.05), while triglyceride levels were decreased in LDM and psoas major muscle by melatonin treatment (P < 0.05). Transcriptome sequencing showed melatonin induced the expression of genes related to skeletal muscle hypertrophy and fatty acid oxidation. Enrichment analysis indicated that melatonin regulated cholesterol metabolism, protein digestion and absorption, and mitophagy signaling pathways in muscle. Gene set enrichment analysis also confirmed the effects of melatonin on skeletal muscle development and mitochondrial structure and function. Moreover, quantitative real-time polymerase chain reaction analysis revealed that melatonin supplementation elevated the gene expression of cell differentiation and muscle fiber development, including paired box 7 (PAX7), myogenin (MYOG), myosin heavy chain (MYHC) IIA and MYHC IIB (P < 0.05), which was accompanied by increased insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 5 (IGFBP5) expression in LDM (P < 0.05). Additionally, melatonin regulated lipid metabolism and activated mitochondrial function in muscle by increasing the mRNA abundance of cytochrome c oxidase subunit 6A (COX6A), COX5B, and carnitine palmitoyltransferase 2 (CPT2) and decreasing the mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), acetyl-CoA carboxylase (ACC) and fatty acid-binding protein 4 (FABP4) (P < 0.05). Together, our results suggest that melatonin could promote skeletal muscle growth and muscle fiber hypertrophy, improve mitochondrial function and decrease fat deposition in muscle.

Due to its extensive biological functions, melatonin has been widely used in animal production in recent years. The purpose of this study was to investigate the effects of melatonin on growth performance, muscle development, and lipid metabolism of weaned piglets. Twelve 28-d-old DLY (Duroc × Landrace × Yorkshire) weaned piglets were randomly divided into two groups: control group and melatonin group. The results showed that melatonin supplementation daily had no effect on growth performance, but increased muscle weight, eye muscle area, and decreased the liver weight in weaned piglets. Consistently, the cross-sectional area of myofiber increased, while triglyceride levels decreased in muscle. Melatonin induced the expression of genes related to skeletal muscle hypertrophy and fatty acid oxidation in muscle through transcriptome sequencing. Additionally, melatonin regulated cholesterol metabolism, protein digestion and absorption, and mitophagy signaling pathways in muscle. Gene set enrichment analysis also confirmed the effects of melatonin on skeletal muscle development and mitochondrial function. Moreover, melatonin supplementation elevated the gene expression of cell differentiation and muscle fiber development. Additionally, melatonin inhibited the mRNA expression related to fat synthesis while improved mitochondrial function in muscle. Together, our results suggest melatonin could promote skeletal muscle growth and muscle fiber hypertrophy, enhance mitochondrial function and decrease fat deposition in muscle.

Purification methods to reduce interference by dextran sodium sulfate with quantification of gene expression in intestinal tissue samples from a piglet model of colitis.

Dextran sodium sulfate (DSS) is commonly used to induce intestinal (i.e., colonic) inflammation in a variety of animal models. However, DSS is known to cause interference when using quantitative-real time polymerase chain reaction (qRT-PCR) methods, thereby invalidating accurate and precise measurement of tissue gene expression. Therefore, the goal of this study was to determine whether different mRNA purification methods would reduce DSS-interference. Colonic tissue samples were collected at postnatal days (PND) 27 or 28 from pigs that had not been administered DSS (Control), and two independent groups of pigs that received 1.25 g of DSS/kg of BW/d (DSS-1 and DSS-2) from PND 14 to 18. Tissue samples collected were subsequently stratified into three purification methods (i.e., 9 total treatment × method combinations), including: 1) no purification, 2) purification with lithium chloride (LiCl), or 3) purification using spin column filtration. All data were analyzed using a one-way ANOVA in the Mixed procedure of SAS. The average RNA concentrations across all treatments were between 1,300 and 1,800 µg/µL for all three in vivo groups. Although there were statistical differences among purification methods, the 260/280 and 260/230 ratios fell between acceptable limits of 2.0 to 2.1 and 2.0 to 2.2, respectively, for all treatment groups. This confirms the RNA quality was adequate and not influenced by purification method in addition to suggesting the absence of phenol, salts, and carbohydrate contamination. For pigs in the Control group that did not receive DSS, qRT-PCR Ct values of four cytokines were achieved, though these values were not altered by purification method. For pigs that had undergone DSS dosing, those tissues subjected to either no purification or purification using LiCl did not generate applicable Ct values. However, when tissues derive from DSS-treated pigs underwent spin column purification, half of the samples from DSS-1 and DSS-2 groups generated appropriate Ct estimates. Therefore, spin column purification appeared to be more effective than LiCl purification, but no method was 100% effective, so caution should be exercised when interpreting gene expression results from studies where animals are exposed to DSS-induced colitis.

Dextran sodium sulfate (DSS) is a chemical used to experimentally induce colonic inflammation in animal models. However, DSS causes chemical inhibition of processes involved with quantitative real-time polymerization chain reaction, thereby inhibiting the measurement of gene expression in tissues. In this study, differing methods of RNA purification were applied to remove DSS inhibition. Because no purification methods were 100% effective in alleviating this interference, caution should be exercised when interpreting gene expression results from studies where animals are exposed to DSS-induced colitis.

Plant-oriented microbiome inoculum modulates age-related maturation of gut-mucosal expression of innate immune and barrier function genes in suckling and weaned piglets.

In the immediate time after weaning, piglets often show symptoms of gut inflammation. The change to a plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite profile in digesta may be causative factors for the observed inflammation. We used the intestinal loop perfusion assay (ILPA) to investigate jejunal and colonic expression of genes for antimicrobial secretion, oxidative stress, barrier function, and inflammatory signaling in suckling and weaned piglets when exposed to "plant-oriented" microbiome (POM) representing postweaning digesta with gut-site specific microbial and metabolite composition. Two serial ILPA were performed in two replicate batches, with 16 piglets preweaning (days 24 to 27) and 16 piglets postweaning (days 38 to 41). Two jejunal and colonic loops were perfused with Krebs-Henseleit buffer (control) or with the respective POM for 2 h. Afterward, RNA was isolated from the loop tissue to determine the relative gene expression. Age-related effects in jejunum included higher expression of genes for antimicrobial secretions and barrier function as well as reduced expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Age-related effects in the colon comprised downregulation of the expression of pattern-recognition receptors post- compared to preweaning (P < 0.05). Likewise, age reduced the colonic expression of genes encoding for cytokines, antimicrobial secretions, antioxidant enzymes, and tight-junction proteins post- compared to preweaning. Effect of POM in the jejunum comprised an increased the expression of toll-like receptors compared to the control (P < 0.05), demonstrating a specific response to microbial antigens. Similarly, POM administration upregulated the jejunal expression of antioxidant enzymes (P < 0.05). The POM perfusion strongly upregulated the colonic expression of cytokines and altered the expression of barrier function genes, fatty acid receptors and transporters, and antimicrobial secretions (P < 0.05). In conclusion, results indicated that POM signaled via altering the expression of pattern-recognition receptors in the jejunum, which in turn activated the secretory defense and decreased mucosal permeability. In the colon, POM may have acted pro-inflammatory via upregulated cytokine expression. Results are valuable for the formulation of transition feeds for the immediate time after weaning to maintain mucosal immune tolerance towards the novel digesta composition.

After weaning, piglets often show symptoms of gut inflammation and reduced performance. The plant-based diet, lack of sow milk, and the resulting novel gut microbiome and metabolite composition in digesta may be causative. However, the acute response of the gut mucosa when exposed to the novel digesta composition has not been fully elucidated. Here, we used the intestinal loop perfusion assay to characterize the immediate effect of a plant-oriented microbiome inoculum (POM) representing postweaning digesta composition on gene expression related to innate immune pathways and barrier function at the jejunal and colonic mucosa in suckling and weaned piglets. Results showed that the recognition of microbial components and barrier function changed in the jejunal and colonic mucosa from pre- to postweaning, indicating age-related maturation and priming by digesta compounds prior to the intestinal loop perfusion assay. In the jejunum, exposure to POM increased expression of receptors recognizing microbial components. In the colon, POM exposure upregulated the expression of genes for pro-inflammatory cytokines and other components of the first line of defense. Results have implications for the formulation of transition feeds for the immediate time after weaning. Inclusion of bioactive porcine milk components may help maintain mucosal immune tolerance towards the novel digesta composition.

Preferential deposition of dairy derived fatty acids in muscle tissue is partially due to the upregulation of CD36 in a low-birth-weight swine model.

Metabolic syndrome is a worldwide health issue. Previous research has revealed that low-birth weight (LBW) swine fed a high-fat (HF) diet were susceptible to insulin resistance (IR) and developed a preferential intestinal lipid absorption, hypertriglyceridemia, and muscle steatosis. We hypothesized that fatty acid transporters such as CD36, FATP4, and FABP2 could potentially explain the development of these conditions. In addition, dairy-derived fatty acids have been shown to be valid biomarkers to assess dairy intake, which can be utilized to investigate muscle lipid deposition in LBW swine. The overall aim of this study was to delineate molecular transport candidates responsible for intestinal lipid absorption and muscle lipid deposition in LBW swine; and secondly to determine what dietary fatty acids might accumulate preferentially in pork muscle when consuming dairy products. At 5 weeks of age, normal birth weight (NBW) and LBW piglets were randomly assigned to three experimental diets: 1-chow diet, 2-HF diet, or 3-isocaloric HF diet supplemented with full fat dairy products. At 12 weeks of age, piglets were euthanized, and carcass, fasting plasma, biceps femoris and jejunum mucosal scrapings were collected. Results showed that HF-fed LBW swine exhibited early signs of IR (fasting glucose, P < 0.05; fasting insulin, P = 0.091; HOMA-IR, P = 0.086) compared with NBW-Chow, which were attenuated with increased dairy intake. Muscle samples from HF-fed LBW swine contained significantly more triglyceride compared to Chow-fed NBW swine (P < 0.05). Increased dairy intake significantly increased myristic acid (C14:0) and DPA (C22:5n3) relative to HF feeding alone (P < 0.05). All HF-fed LBW swine (regardless of dairy intake) exhibited an upregulation of CD36 expression (but not FABP2) compared with NBW littermates in both the small intestine and muscle (P < 0.05). Interestingly, increased dairy intake significantly increased the Canadian Lean Yield percentage in LBW swine fed an HF diet (P < 0.05). Findings from this study provide evidence on the mechanistic pathway of intestinal and muscle lipid metabolism in an innovative LBW swine model. We have also revealed that increasing dairy intake can enhance the incorporation of dietary long-chain polyunsaturated fatty acids into pork, as well as increasing the predicted lean yield of the carcass.

Metabolic syndrome affects millions of people worldwide, and large animal models represent a unique opportunity for research advancement. Intensive swine production can induce low-birth weight (LBW) litters. We have developed an innovative LBW swine model to investigate insulin resistance and dyslipidemia. We present evidence to explain how LBW swine can upregulate lipid intestinal absorption as well as preferentially increase pork marbling. We have also identified a potential added value approach to increase healthy fatty acids in pork and/or increase the carcass lean yield in LBW swine.