Enhancement of porcine in vitro embryonic development through luteolin‑mediated activation of the Nrf2/Keap1 signaling pathway

Background Oxidative stress,caused by an imbalance in the production and elimination of intracellular reactive oxygen species(ROS),has been recognized for its detrimental effects on mammalian embryonic development.Luteolin(Lut)has been documented for its protective effects against oxidative stress in various studies.However,its specific role in embryonic development remains unexplored.This study aims to investigate the influence of Lut on porcine embryonic development and to elucidate the underlying mechanism.Results After undergoing parthenogenetic activation(PA)or in vitro fertilization,embryos supplemented with 0.5μmol/L Lut displayed a significant enhancement in cleavage and blastocyst formation rates,with an increase in total cell numbers and a decrease in the apoptosis rate compared to the control.Measurements on D2 and D6 revealed that embryos with Lut supplementation had lower ROS levels and higher glutathione levels compared to the control.Moreover,Lut supplementation significantly augmented mitochondrial content and membrane potential.Intriguingly,activation of the Nrf2/Keap1 signaling pathway was observed in embryos supplemented with Lut,leading to the upregulation of antioxidant-related gene transcription levels.To further validate the relationship between the Nrf2/Keap1 signaling pathway and effects of Lut in porcine embryonic development,we cultured PA embryos in a medium supplemented with brusatol,with or without the inclusion of Lut.The positive effects of Lut on developmental competence were negated by brusatol treatment.Conclusions Our findings indicate that Lut-mediated activation of the Nrf2/Keap1 signaling pathway contributes to the enhanced production of porcine embryos with high developmental competence,and offers insight into the mechanisms regulating early embryonic development.

Porcine enteric alphacoronavirus infection increases lipid droplet accumulation to facilitate the virus replication

Coronaviruses are widely transmissible between humans and animals, causing diseases of varying severity. Porcine enteric alphacoronavirus(PEAV) is a newly-discovered pathogenic porcine enteric coronavirus in recent years, which causes watery diarrhea in newborn piglets. The host inflammatory responses to PEAV and its metabolic regulation mechanisms remain unclear, and no antiviral studies have been reported. Therefore, we investigated the pathogenic mechanism and antiviral drugs of PEAV. The transcriptomic analysis of PEAV-infected host cells revealed that PEAV could upregulate lipid metabolism pathways. In lipid metabolism, steady-state energy processes, which can be mediated by lipid droplets(LDs), are the main functions of organelles. LDs are also important in viral infection and inflammation. In infected cells, PEAV increased LD accumulation, upregulated NF-κB signaling, promoted the production of the inflammatory cytokines IL-1β and IL-8, and induced cell death. Inhibiting LD accumulation with a DGAT-1 inhibitor significantly inhibited PEAV replication, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. The NF-κB signaling pathway inhibitor BAY11-7082 significantly inhibited LD accumulation and PEAV replication. Metformin hydrochloride also exerted anti-PEAV effects and significantly inhibited LD accumulation, downregulated the NF-κB signaling pathway, reduced the production of IL-1β and IL-8, and inhibited cell death. LD accumulation in the lipid metabolism pathway therefore plays an important role in the replication and pathogenesis of PEAV, and metformin hydrochloride inhibits LD accumulation and the inflammatory response to exert anti-PEAV activity and reducing pathological injury. These findings contribute new targets for developing treatments for PEAV infections.

Intraocular pressure before and after capsulorhexis using two viscoelastic substances and two surgical approaches in enucleated porcine eyes

AIM:To investigate the influence of ophthalmic viscoelastic devices(OVDs)and different surgical approaches on the intraocular pressure(IOP)before and after creation of the curvilinear circular capsulorhexis(CCC)as a measure for anterior chamber stability during this maneuver.METHODS:Prospective experimental WetLab study carried out on enucleated porcine eyes.IOP was measured before and after CCC with the iCare Rebound tonometer(iCare ic200;iCare Finland Oy,Vantaa,Finland).The OVDs used were a cohesive one[Z-Hyalin,Carl Zeiss Meditec AG,Germany;hyaluronic acid(HA)]and a dispersive[Z-Celcoat,Carl Zeiss Meditec AG,Germany;hydroxy propylmethylcellulosis(HPMC)].The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs.RESULTS:Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC.The use of different OVDs made no difference.Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg.The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used.CONCLUSION:Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes.The use of different OVD has no significant influence on IOP drop.

Small-diameter acellular porcine corneal stroma for peripheral corneal ulceration treatment

AIM:To evaluate the clinical efficacy of small-diameter acellular porcine corneal stroma(SAPS)for the treatment of peripheral corneal ulceration(PCU).METHODS:This retrospective clinical study included 18 patients(18 eyes)with PCU between April 2018 and December 2020.All patients had PCU and underwent lamellar keratoplasty with SAPS.Observation indicators included preoperative and postoperative best-corrected visual acuity(BCVA)and transparency of SAPS.The infection control rate in the surgical eye-lesion area was also calculated.RESULTS:Eighteen patients underwent lamellar keratoplasty with SAPS to treat PCU.None of the patients experienced rejection after 6mo(18/18)and 12mo(16/16)of follow-up.The BCVA(0.47±0.30)at the 6mo followup after operation was significantly improved compared with the baseline(0.99±0.80),and the difference was statistically significant(Z=-3.415,P<0.05).The BCVA at the 12mo follow-up after operation was not statistically significant compared to the 6mo(Z=0,P=1).With time,the SAPS graft gradually became transparent.At the 6mo(18/18)and 12mo(16/16)follow-up,none of the patients had recurrent corneal infection.CONCLUSION:SAPS is clinically effective in the treatment of PCU,improving the patient’s BCVA and reducing the incidence of rejection after keratoplasty.

Isorhamnetin protects porcine oocytes from zearalenone-induced reproductive toxicity through the PI3K/Akt signaling pathway

Background Zearalenone(ZEA)widely exists in moldy grains,which seriously destroys the fertility of females.Isorhamnetin,a natural flavonoid,has extensive of pharmacological activities.However,the beneficial effect and the underlying molecular mechanism of isorhamnetin involvement in ZEA-induced porcine oocyte damage have not been investigated.Methods Oocytes were treated with different concentrations of ZEA(3,5,8 and 10μmol/L)and isorhamnetin(5,10,20 and 30μmol/L)for 44 h at 39℃.ZEA(5μmol/L)and isorhamnetin(10μmol/L)were selected for subsequent studies.Polar body exclusion rate,apoptosis rate and apoptosis related proteins,ROS levels and SOD2 protein,mitochondrial membrane potential and distribution,endoplasmic reticulum distribution and proteins expression,and PI3K,Akt and p-Akt proteins expression of oocytes were detected.In addition,the effect of PI3K antagonist(LY294002)on oocyte nuclear maturation and apoptosis were used to determine the involvement of PI3K/Akt signaling pathway.Results Our findings showed that ZEA exposure damaged oocytes and isorhamnetin therapy restored the developmental capability of porcine oocytes.Isorhamnetin promoted polar body extrusion rate to rescue ZEA-induced meiotic arrest in porcine oocytes.Isorhamnetin alleviated ZEA-induced oxidative stress by stimulating SOD2 protein expression and inhibiting ROS production.Moreover,isorhamnetin enhanced normal mitochondrial distribution and mitochondrial membrane potential to prevent mitochondrial dysfunction induced by ZEA.Changing the expression of endoplasmic reticulum stress-related marker proteins(CHOP,GRP78)and the distribution rate of normal endoplasmic reticulum showed that isorhamnetin relieved ZEA-caused endoplasmic reticulum stress.Mechanistically,isorhamnetin decreased Bax/Bcl-2 protein expression and inhibited ZEA-induced apoptosis through PI3K/Akt signaling pathway.Conclusions Collectively,these results suggest that isorhamnetin protects oocytes from ZEA-caused damage through PI3K/Akt signaling pathway,which enhances meiotic maturation and mitochondrial function,and inhibits early apoptosis,oxidative stress and endoplasmic reticulum stress in porcine oocytes.Our study provides a new strategy for solving the reproductive toxicity induced by ZEA and treating woman infertility.

Pig macrophages with site-specific edited CD163 decrease the susceptibility to infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome(PRRS)is recognized as one of the most infectious viral diseases of swine.Although Cluster of differentiation 163(CD163)is identified as an essential receptor for mediating PRRS virus(PRRSV)infection,the important residues involved in infection on CD163 are still unclear.Therefore,it is very important to identify these key residues to study the mechanism of PRRSV infection and to generate anti-PRRSV pigs.In this study,we first generated immortalized porcine alveolar macrophage(IPAM)cell lines harboring 40-residues(residues 523-562,including R561(arginine(R)at position 561))deletion of CD163.PRRSV infection experiments showed that these IPAM cell lines were completely resistant to PRRSV infection.We then generated cloned pigs carrying CD163-R561A(an arginine(R)to alanine(A)substitution at position 561 of CD163).PRRSV challenge experiments in porcine alveolar macrophages(PAMs)isolated from the CD163-R561A pigs showed significantly lower susceptibility to PRRSV than that of CD163-R561 PAMs.Through this study,we show that CD163523-562 contains essential residues for mediating PRRSV infection,and that CD163 R561 significantly contributes to PRRSV infection but is not essential for infection.These functional sites can therefore serve as new targets for understanding the mechanism of PRRSV infection.Furthermore,CD163-R561A pigs can be used as an important model for improving pig germplasm with resistance against PRRSV.

Anethole improves the developmental competence of porcine embryos by reducing oxidative stress via the sonic hedgehog signaling pathway

Background Anethole(AN)is an organic antioxidant compound with a benzene ring and is expected to have a positive impact on early embryogenesis in mammals.However,no study has examined the effect of AN on porcine embryonic development.Therefore,we investigated the effect of AN on the development of porcine embryos and the underlying mechanism.Results We cultured porcine in vitro-fertilized embryos in medium with AN(0,0.3,0.5,and 1 mg/mL)for 6 d.AN at 0.5 mg/mL significantly increased the blastocyst formation rate,trophectoderm cell number,and cellular survival rate compared to the control.AN-supplemented embryos exhibited significantly lower reactive oxygen species levels and higher glutathione levels than the control.Moreover,AN significantly improved the quantity of mitochondria and mitochondrial membrane potential,and increased the lipid droplet,fatty acid,and ATP levels.Interestingly,the levels of proteins and genes related to the sonic hedgehog(SHH)signaling pathway were significantly increased by AN.Conclusions These results revealed that AN improved the developmental competence of porcine preimplantation embryos by activating SHH signaling against oxidative stress and could be used for large-scale production of high-quality porcine embryos.

Dynamic compressive response of porcine muscle measured using a split Hopkinson bar system with a pair of PVDF force transducers

The classic metallic Split Hopkinson Pressure Bar(SHPB)cannot capture the transmitted signal accurately when measuring soft biological tissue,because of the very low wave impedance and strength of this material.So the dynamic compressive response of porcine muscle has been investigated by using a modified SHPB.The forces on both ends of the sample measured using Polyvinylidene fluor(PVDF)transducers were applied to calculate the stress in the specimen instead of the strain gauge signal on the transmitted bar.Moreover,a circular cardboard disk pulse shaper was applied for generating a suitable incident pulse to achieve stress equilibrium and constant strain rates in the specimens.Then,the dynamic mechanical properties of porcine muscle parallel and perpendicular to the fiber directions were measured,and the stress equilibrium process during loading was analyzed,as well as the inertia-induced extra stress being corrected.Furthermore,quasi-static tests were conducted at two different strain rates to investigate the strain rate dependence using a universal material testing machine.The results show that the stress-strain curves are sensitive to strain rate in the two different loading directions.The compressive stress perpendicular to the fiber direction is stiffer than that parallel to the fiber direction.In addition,a strain rate-dependent constitutive model was developed based on the mechanical response of the muscle at different strain rates and fitted to the experimental data.The results show that the overall fit is good,and the constitutive model could describe the muscle's dynamic mechanical properties.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus,African swine fever virus,and atypical porcine pestivirus

With the implementation of the C-strain vaccine,classical swine fever(CSF) has been under control in China,which is currently in a chronic atypical epidemic situation.African swine fever(ASF) emerged in China in 2018 and spread quickly across the country.It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety.Atypical porcine pestivirus(APPV) was first detected in Guangdong Province,China,in 2016,which mainly harms piglets and has a local epidemic situation in southern China.These three diseases have similar clinical symptoms in pig herds,which cause considerable losses to the pig industry.They are difficult to be distinguished only by clinical diagnosis.Therefore,developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential.In this study,three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV(5’ UTR),African swine fever virus(ASFV)(B646L),and APPV(5’ UTR),followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay.The results showed that the method did not cross-react with other swine pathogens(porcine circovirus type 2(PCV2),porcine reproductive and respiratory syndrome virus(PRRSV),foot-and-mouth disease virus(FMDV),pseudorabies virus(PRV),porcine parvovirus(PPV),and bovine viral diarrhea virus BVDV).The sensitivity results showed that CSFV,ASFV,and APPV could be detected as low as 1 copy μL–1;the repeatability results showed that the intra-assay and interassay coefficient of variation of ASFV,CSFV,and APPV was less than 1%.Twenty-two virus samples were detected by the multiplex real-time PCR,compared with national standard diagnostic and patented method assay for CSF(GB/T 27540–2011),ASF(GB/T 18648–2020),and APPV(CN108611442A),respectively.The sensitivity of this triple real-time PCR for CSFV,ASFV,and APPV was almost the same,and the compliance results were the same(100%).A total of 451 clinical samples were detected,and the results showed that the positive rates of CSFV,ASFV,and APPV were 0.22% (1/451),1.3%(6/451),and 0%(0/451),respectively.This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV,ASFV,and APPV.

Associations between maternal vitamin D status and porcine litter characteristics throughout gestation

Emerging evidence suggests an important role of vitamin D in the establishment and maintenance of pregnancy,and the regulation of foetal growth across mammalian species.However,the temporal changes in maternal vitamin D sta-tus throughout gestation in the pig and the relationship between maternal vitamin D status and litter characteristics of interest across gestation remain poorly understood and under-investigated.The abundance of 25(OH)D in maternal plasma was quantified by HPLC–MS/MS at gestational days(GD)18,30,45,60 and 90(n=5–11 gilts/GD).Maternal plasma 25(OH)D concentrations significantly increased between GD18 and GD30(P<0.05).The relationship between maternal vitamin D metabolite concentrations and litter characteristics of interest including gilt weight,ovulation rate,mean litter weight,number of live foetuses,percentage prenatal survival,and sex ratio of the litter was assessed.Maternal 25(OH)D(P=0.059)concentrations tended to be positively associated with percentage prenatal survival on GD60.On GD90,maternal 25(OH)D(P<0.05)concentrations were inversely associated with gilt weight.Maternal plasma 25(OH)D concentrations were inversely associated with the percentage of male foetuses in the litter on GD90(P<0.05).This study has provided novel insights into temporal changes in maternal vitamin D status throughout ges-tation and the relationship between maternal vitamin D status and the economically important litter characteristics of gilt weight,percentage prenatal survival and percentage of male foetuses in the litter.Improving the understanding of the role of vitamin D across important developmental timepoints in relation to foetal growth is essential to improve reproductive success in livestock species.

Comparison of emergency surgical cricothyroidotomy and percutaneous cricothyroidotomy by experienced airway providers in an obese,in vivo porcine hemorrhage airway model

Background: Emergency front-of-neck airway(eFONA) is a life-saving procedure in “cannot intubate, cannot oxygenate”(CICO). The fastest and most reliable method of eFONA has not been determined. We compared two of the most advocated approaches: surgical cricothyroidotomy and percutaneous cricothyroidotomy, in an obese, in vivo porcine hemorrhage model, designed to introduce real-time physiological feedback, relevant and high provider stress. The primary aim was to determine the fastest method to secure airway. Secondary aims were arterial saturation and partial pressure of oxygen, proxy survival and influence of experience.Methods: Twelve pigs [(60.3±4.1) kg] were anesthetized and exposed to 25%–35% total blood volume hemorrhage before extubation and randomization to Seldinger technique “percutaneous cricothyroidotomy”(n=6) or scalpelbougie-tube technique “surgical cricothyroidotomy”(n=6). Specialists in anesthesia and intensive care in a tertiary referral hospital performed the eFONA, simulating an actual CICO-situation.Results: In surgical cricothyroidotomy vs. percutaneous cricothyroidotomy, the median(interquartile range, IQR) times to secure airway were 109(IQR 71–130) s and 298(IQR 128–360) s(P=0.0152), arterial blood saturation(SaO2) were 74.7(IQR 46.6–84.2)% and 7.9(IQR 4.1–15.6)%(P=0.0167), PaO_(2) were 7.0(IQR 4.7–7.7) kPa and 2.0(IQR 1.1–2.9) kPa(P=0.0667), and times of cardiac arrest(proxy survival) were 137–233 s, 190(IQR 143–229) s, from CICO. All six animals survived surgical cricothyroidotomy, and two of six(33%) animals survived percutaneous cricothyroidotomy. Years in anesthesia, 13.5(IQR 7.5–21.3), did not influence time to secure airway.Conclusions: eFONA by surgical cricothyroidotomy was faster and had increased oxygenation and survival, when performed under stress by board certified anesthesiologists, and may be an indication of preferred method in situations with hemorrhage and CICO, in obese patients.

Influence of Hypericum perforatum Extract on Piglet Infected with Porcine Respiratory and Reproductive Syndrome Virus

To study the influence of Hypericum perforatum extract （HPE） on piglets infected with porcine respiratory and reproductive syndrome virus （PRRSV）, enzyme-labeled immunosorbent assay （ELISA） and cytopathic effect （CPE） were used to determine in vitro whether HPE could induce swine pulmonary alveolar macrophages （PAMs） to secrete IFN-γ, and whether PRRSV titers in PAMs were affected by the levels of HPE-induced IFN-γ. HPE （200 mg·kg-1） was administrated by oral gavage to piglets infected with the PRRSV in vivo to observe whether HPE affected the viremia, lung viral titers, and weight gain of piglets infected with PRRSV. The results showed that HPE was capable of inducing PAMs to produce IFN-γ in a dose dependent manner and HPE pretreatment was capable of significantly reducing PRRSV viral titers in PAMs （P〈 0.01）. Administration of HPE to the PRRSV-infected animals significantly （P〈 0.05） reduced viremia over time as compared with the PRRSV-infected animals. But there was not significant decrease in lung viral titers at day 21 post-infection between the HPE- treated animals and the PRRSV-infected control piglets. There were no significant differences in weight gain over time among the HPE-treatment animals, the normal control, and the HPE control animals. The PRRSV-infected animals caused significant （P〈 0.01） growth retardation as compared with the HPE controls and the normal piglets. It suggested that HPE might be an effective novel therapeutic approach to diminish the PRRSV-induced disease in swine.

Genetic analysis of reproductive performance in sows during porcine reproductive and respiratory syndrome(PRRS)and porcine epidemic diarrhea(PED)outbreaks

Background: Porcine reproductive and respiratory syndrome(PRRS) is one of the most infectious swine diseases in the world, resulting in over 600 million dollars of economic loss in the USA alone. More recently, the USA swine industry has been having additional major economic losses due to the spread of porcine epidemic diarrhea(PED).However, information regarding the amount of genetic variation for response to diseases in reproductive sows is still very limited. The objectives of this study were to identify periods of infection with of PRRS virus(PRRSV) and/or PED virus(PEDV), and to estimate the impact their impact on the phenotypic and genetic reproductive performance of commercial sows.Results: Disease(PRRS or PED) was significant(P < 0.05) for all traits analyzed except for total piglets born.Heritability estimates for traits during Clean(without any disease), PRRS, and PED ranged from 0.01(number of mummies;Clean and PED) to 0.41(abortion;PED). Genetic correlations between traits within disease statuses ranged from-0.99(proportion born dead with number weaned;PRRS) to 0.99(number born dead with born alive;Clean). Within trait, between disease statuses, estimates ranged from-0.17(number weaned between PRRS and PED) to 0.99(abortion between Clean and PRRS).Conclusion: Results indicate that selection for improved performance during PRRS and PED in commercial sows is possible and would not negatively impact performance in Clean environments.

Expression of Porcine Interleukin-2 and Porcine Interleukin-6 and Their Adjuvant Effects on Gene Deleted Vaccine of Pseudorabies Virus(TK^-/gG^-/LacZ^+)

Porcine interleukin-2 and porcine interleukin-6 cDNA sequences were cloned into the expressing vectors pET-28a and pGEX-KG respectively. They were expressed in E. coli BL21(DE3)with high-level production. The gene deleted vaccine of pseudorabies virus Ea strain(TK-/gG-/LacZ+)was mixed with the two different purified recombinant proteins each, or both, with the doses of 2, 5 or 10 μg ml-1. Ten groups of pseudorabies negative antibody swines were immuned twice with tested vaccines with different doses, or control vaccine, respectively. The antibody liters of the test groups were detected by neutralization test, and the daily weight gains of swines were calculated and analyzed statistically. In the study, all the neutralizing antibody ti-ters in test groups were higher than the control group, and the recombinant proteins appeared a dose dependent adjuvant effect. The tested vaccines with 2 μg ml-1 pIL-2 and with 10 μg ml-1 pIL-2/pIL-6 got significant and extremely significant differences, compared with the vaccines without pILs. The difference of the daily weight gain indicated the potential positive influence of pIL-2 and pIL-6 on immune protection.

Expression of Core Domain of Porcine Zona Pellucida 3β in E.coli

To obtain the recombinant core domain of porcine zone pellucida 3β （cZP3β） for the further research on its functions Methods The nucleotide sequence region from 44 to 306 codons of pZP3β entire eDNA was obtained by PCR and then was cloned into pET-3c vector. After being identified, recon was transformed into E.coli BL21 （DE3） pLysS and then induced by IPTG. Results The recombinant cZP3β was expressed in E. coli up to 15% of total cellular proteins, and was made sure by Western blot analysis. Conclusion The research on expression of core domain of pZP3β could benefit to further investigation of its immunogenicity and the development of antigen preparation.

Porcine NF-κB p65 Subunit:Molecular Characterization,Tissue Expression and Transcriptional Profile in Porcine Epidemic Diarrhea Virus-infected IPEC-J2 Cells

The p65 protein is a functional subunit of NF-κB family and exhibits a crucial role in host immune and inflammatory responses,apoptosis and tumor proliferation if improperly-regulated.Given its ubiquitous association with nearly all the animal cells and its pleotropic functions,the gene encoding NF-κB p65 subunit was cloned and sequenced from porcine kidney(PK-15)cells.The gene was 1662 bp in length,encoded a 553-amino acid protein and contained the prototypical NF-κB functional domains.Real-time quantitative RT-PCR and Western blot were used to characterize the transcription and expression levels of the p65 in different pig tissues.The results indicated that the p65 gene and protein were both broadly expressed in pig tissues,but most highly expressed in the intestine-associated lymph nodes and the lungs.To localize the recombinant protein in intestinal porcine epithelial cells(IPEC-J2),the gene was subcloned into the vector pEGFP(pEGFP-p65).Using fluorescence microscopy,the protein was found confined to the cytoplasm in normal cells;however,during porcine epidemic diarrhea virus(PEDV)infection,mRNA and protein expression were significantly up-regulated and the protein exhibited an overt tendency for nuclear translocalization consistent with a regulatory role in antiviral innate immunity.

Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan Province

[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.

氨基化大孔硅胶共价固定Pancreas porcine脂肪酶的研究

本研究以大孔硅胶为材料,对其进行环氧基和氨基两步活化,然后利用碳化二亚胺(EDC)将氨基化硅胶与Pancreas porcine脂肪酶(PPL)进行共价固定化。通过优化固定化条件,在EDC与蛋白摩尔比为50:1;载体与粗酶质量比为10:1,固定化pH为6.0,固定化时间为8 h的条件下获得最大固定化酶酶活为86.67 U/g,远远高于未修饰硅胶物理吸附固定PPL所得固定化酶酶活(33.33U/g)。此外,本文还利用扫描电镜(SEM)和傅立叶变换光谱仪(FT-IR)对固定化前后硅胶进行了结构表征,固定化后硅胶表面出现明显的丝状蛋白,且固定化酶在1543 cm-1处出现酰胺Ⅱ带,说明PPL成功共价固定于氨基化硅胶上。通过对所得固定化酶性质进行分析,结果表明,固定化酶最适反应pH向碱偏移一个单位,最适反应温度提高了5℃,热稳定性明显提高。

Host immune response to infection with porcine circoviruses

Porcine circovirus type 2(PCV2),which serves as a major causative agent of PCV2-associated diseases and causes severe loss to the pig industry worldwide,can dysregulate the immune response and induce immunosuppression in PCV2-infected pigs.Similar to PCV2(porcine circovirus type 3(PCV3),a newly identified swine circovirus which might be closely associated with porcine dermatitis and nephropathy syndrome,reproductive disorder,and multisystemic inflammatoty responses,also interferes with host immune defense.Interaction between host immune system and PCVs is considered to be a crucial determinant of pathogenicity in pigs.Here,we sought to briefly discuss the current knowledge regarding the interaction of porcine circovirus type 2 and/or 3 with host immune cells and immune responses to better depict the viral immunomodulatory capacity,pathogenic mechanisms,and the future research direction in host immune responses to infection with PCV2 and PCV3.

Primary porcine hepatocytes with portal vein serum cultured on microcarriers or in spheroidal aggregates

AIM To develop a culture mode providingdurable biomaterials with high yields andactivities used in bioartificial liver.METHODS Hepatocytes were isolated from awhole pig liver by Seglen’s method of orthotopicperfusion with collagenase.In culture onmicrocarriers,primary porcine hepatocyteswere inoculated at a concentration of 5×10~7/mLinto the static culture systems containing 2 g/LCytodex-3,then supplemented with 100 mL/Lfetal calf serum(FCS)or 100 mL/L porcineportal vein serum(PPVS)respectively.Inspheroidal aggregate culture hepatocytes wereinoculated into 100 mL siliconized flasks at aconcentration of 5.0×10~6/mL.RESULTS In culture on microcarriershepatocytes tended to aggregate on Cytodex-3obviously after being inoculated.Typical multi-cellular aggregated spheroids could be found inthe two systems 24 h-48 h after hepatocyteswere cultured.The morphological charact-eristics and synthetic functions were maintainedfor 5 wk in FCS culture system and 8 wk in PPVSculture system.In spheroidal aggregate cultureabout 80%-90% isolated hepatocytes becameaggregated spheroids 24h after cultured insuspension and mean diameter of the spheroidswas 100μm.The relationship among thehepatocytes resembled that in the liver in vivo.Synthetic functions of albumin and urea of the spheroids were twice those of hepatocytescultured on monolayers.CONCLUSION As high-yields and high-activitymodes of culture on microcarriers or inspheroidal aggregate culture with portal veinserum are promising to provide biomaterials forbioartificial liver(BAL)efficiently.