Pyrroloquinoline quinone regulates the redox status in vitro and in vivo of weaned pigs via the Nrf2/HO-1 pathway

Background:Oxidative stress is a main cause of piglet gut damage and diarrhea.Pyrroloquinoline quinone(PQQ),is a novel redox cofactor with antioxidant properties.However,the effect and mechanism that PQQ supplementation decreases oxidative injury in weaned pigs is not understood.Therefore,the aim of this study is to confirm the effect of PQQ on regulating redox status in weaned pigs and the mechanism for antioxidant function by porcine intestinal epithelial cell line(IPEC-J2)challenged with H_(2)O_(2).Results:Experiment 1,144 Duroc×Landrace×Yorkshire pigs(weaned at 28 d)were allocated to four groups:received a basal diet(control)and diets supplemented with 0.15%,0.30%and 0.45%PQQ,respectively.On d 28,growth performance,diarrhea incidence and redox factors were measured.Experiment 2,IPEC-J2 were treated with or without PQQ in the presence or absence of H_(2)O_(2)for indicated time points.Experiment 3,IPEC-J2 were transfected with or without Nrf2 siRNA,then treated according to Experiment 2.The cell viability,redox factors,protein of tight junctions and Nrf2 pathway were determined.In vivo,PQQ supplementation demonstrated dose-related improvements in average daily gain,and gain to feed ratio(Linear P<0.05).During d 0–28,compared to controls,0.45%PQQ supplementation for pigs decreased diarrhea incidence and MDA content in liver and jejunum,and increased concentration of SOD in liver;0.3%PQQ supplementation decreased ileal and liver MDA concentration;and 0.15%PQQ supplementation decreased ileal MDA concentration(P<0.05).In vitro,compared to cells cultured with H_(2)O_(2),pre-treatment with PQQ increased cell viability,tight junction proteins expression including ZO-1,ZO-2,Occludin and Claudin-1;and decreased ROS concentration and level of Caspase-3(P<0.05);as well as upregulated the ratio of Bcl-2 to Bax and protein expression of nuclear Nrf2,HO-1.Notably,Nrf2 knockdown by transfection with Nrf2 siRNA largely abrogated the positive effects of PQQ pretreatment on H_(2)O_(2)-induced intracellular changes.Conclusions:PQQ administration attenuated oxidative stress in weaned pigs which is associated with activation of Nrf2/HO-1 pathway.

Use of near-infrared reflectance spectroscopy for the rapid determination of the digestible energy and metabolizable energy content of corn fed to growing pigs

Background： The ability of near-infrared reflectance spectroscopy（NIRS） to determine the digestible energy（DE）and metabolizable energy（ME） content of corn fed to growing pigs was tested. One hundred and seventeen corn samples, comprising different planting regions and varieties were collected from all over China in a three-year period. The samples were randomly split into a calibration set（n = 88） and a validation set（n = 29）. The actual and calculated DE and ME content of the corn samples was determined by digestion-metabolism experiments and the prediction equations of Noblet and Perez（J Anim Sci. 71：3389–98,1993）. The samples were then subjected to NIRS scanning and calibrations were performed by the modified partial least square（MPLS） regression method based on77 different spectral pre-treatments. The NIRS equations based on the actually determined and calculated DE and ME were built separately and then validated using validation samples.Results： The NIRS equations obtained from actually determined DE, the coefficient of determination for calibration（RSQcal）, cross-validation（R^2CV）, and validation（RSQv） were 0.89, 0.87 and 0.86, and these values for determined ME were 0.87, 0.86 and 0.86. For the NIRS equations built from calculated DE, the RSQcal, R^2CV, and RSQvvalues were 0.88, 0.85 and 0.84, and these values for calculated ME were 0.86, 0.84 and 0.82. Except for the equation based on calculated ME（RPD_v= 2.38, 〈 2.50）, the other three equations built from actually determined energy and calculated DE produced good prediction performance（RPD_vranging from 2.53 to 2.69, 〉 2.50） when applied to validation samples.Conclusion： These results indicate that NIRS can be used as a quantitative method for the rapid determination of the available energy in corn fed to growing pigs, and the NIRS equations based on the actually determined energy produced better predictive performance than those built from calculated energy values.

Effects of Drinking Slightly Acidic Electrolytic Water on Growth Performance and Behavior of Broilers

[Objective]The paper was to study the effects of drinking slightly acidic electrolytic water on growth performance and behavior of Rose 308 broilers,and to provide reference for the application of slightly acidic electrolytic water in broiler breeding.[Method]A total of 300 healthy 10-day-old Rose 308 broilers with similar body weight were randomly divided into five groups,three replicates each group,20 broilers each replicate.The broilers in control group were supplied with normal drinking water,and the broilers in experimental groups consumed slightly acidic electrolytic water with 0.3,0.5,0.7,1.0 mg/L residual chlorine,respectively.The test lasted 21 d.[Result]At 10-30 days of age,the water consumption of broilers in 0.7 and 1.0 mg/L electrolytic water groups were increased by 9.27%and 7.67%respectively compared with the control group(M<0.05).The average daily feed intake(ADFI)of broilers in 0.7 and 1.0 mg/L electrolytic water groups were increased compared with the control group(Q0.05).The average daily gain(ADG)of broilers in 0.7 mg/L electrolytic water group was 11.99%lower than that in control group(M<0.05).The feed gain ratio(F/G)of broilers in 0.5 mg/L electrolytic water group was 12.29%lower than that in control group(M<0.05),and the mortality was the lowest in 0.5 mg/L electrolytic water group.The standing,feeding and drinking frequency of broilers in experimental groups were higher than that in control group,and the flapping behavior of broilers in 0.5 mg/L electrolytic water group was the lowest.[Conclusion]Drinking slightly acidic electrolytic water has positive effect on the growth and behavior of broilers.

A 314-bp SINE insertion in the ZNF2 promoter region may act as a repressor related to regulation of fat deposition in pigs

Retrotransposons,a type of DNA fragment that can mobilize itself on genome,can generate genetic variations and develop for molecular markers based on the insertion polymorphism.Zinc finger proteins(ZNFs)are among the most abundant proteins in eukaryotic animals,and their functions are extraordinarily diverse and particularly important in gene regulation.In the current study,bioinformatic prediction was performed to screen for retrotransposon insertion polymorphisms(RIPs)in six ZNF genes(ZNF2,ZNF3,ZNF7,ZNF8,ZNF10 and ZNF12).Six RIPs in these ZNFs,including one short interspersed nuclear element(SINE)RIP in intron 1 and one long interspersed nuclear element 1(L1)RIP in intron 3 of ZNF2,one SINE RIP in 5′flanking region and one SINE RIP in intron 2 of ZNF3,one SINE RIP in 3′UTR of ZNF7 and one L1 RIP in intron 2 of ZNF12,were discovered and their presence was confirmed by PCR.The impact of the SINE RIP in the first intron of ZNF2,which is close to the core promoter of ZNF2,on the gene activity was investigated by dual-luciferase assay in three cell lines.Our results showed that the SINE insertion in the intron 1 of ZNF2 repressed the core promoter activity extremely significantly(P<0.01)in cervical cancer cells and porcine primary embryonic fibroblasts(HeLa and PEF),thus SINE may act as a repressor.This SINE RIP also significantly(P<0.05)affected the corrected back fat thickness in Yorkshire pigs.The corrected back fat thickness of individuals with SINE insertion in the first intron of ZNF2 was significantly(P<0.05)higher than that of individuals without SINE insertion.In summary,our data suggested that RIPs play important roles in the genetic variations of these ZNF genes and SINE RIP in the intron 1 of ZNF2 may provide a useful molecular marker for the screening of fat deposition in the pig breeding.

Membrane vesicles derived from Streptococcus suis serotype 2 induce cell pyroptosis in endothelial cells via the NLRP3/Caspase-1/GSDMD pathway

Streptococcus suis serotype 2(S.suis 2)is a zoonotic pathogen that clinically causes severe swine and human infections(such as meningitis,endocarditis,and septicemia).In order to cause widespread diseases in different organs,S.suis 2 must colonize the host,break the blood barrier,and cause exaggerated inflammation.In the last few years,most studies have focused on a single virulence factor and its influences on the host.Membrane vesicles(MVs)can be actively secreted into the extracellular environment contributing to bacteria-host interactions.Gram-negative bacteria-derived outer membrane vesicles(OMVs)were recently shown to activate host Caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide(LPS),causing host cell pyroptosis.However,little is known about the effect of the MVs from S.suis 2(Gram-positive bacteria without LPS)on cell pyroptosis.Thus,we investigated the molecular mechanism by which S.suis 2 MVs participate in endothelial cell pyroptosis.In this study,we used proteomics,electron scanning microscopy,fluorescence microscope,Western blotting,and bioassays,to investigate the MVs secreted by S.suis 2.First,we demonstrated that S.suis 2 secreted MVs with an average diameter of 72.04 nm,and 200 proteins in MVs were identified.Then,we showed that MVs were transported to cells via mainly dynamin-dependent endocytosis.The S.suis 2 MVs activated NLRP3/Caspase-1/GSDMD canonical inflammasome signaling pathway,resulting in cell pyroptosis,but it did not activate the Caspase-4/-5 pathway.More importantly,endothelial cells produce large amounts of reactive oxygen species(ROS)and lost their mitochondrial membrane potential under induction by S.suis 2 MVs.The results in this study suggest for the first time that MVs from S.suis 2 were internalized by endothelial cells via mainly dynamin-dependent endocytosis and might promote NLRP3/Caspase-1/GSDMD pathway by mitochondrial damage,which produced mtDNA and ROS under induction,leading to the pyroptosis of endothelial cells.

Repairing whole facial nerve defects with xenogeneic acellular nerve grafts in rhesus monkeys

Acellular nerve allografts conducted via chemical extraction have achieved satisfactory results in bridging whole facial nerve defects clinically,both in terms of branching a single trunk and in connecting multiple branches of an extratemporal segment.However,in the clinical treatment of facial nerve defects,allogeneic donors are limited.In this experiment,we exposed the left trunk and multiple branches of the extratemporal segment in six rhesus monkeys and dissected a gap of 25 mm to construct a monkey model of a whole left nerve defect.Six monkeys were randomly assigned to an autograft group or a xenogeneic acellular nerve graft group.In the autograft group,the 25-mm whole facial nerve defect was immediately bridged using an autogenous ipsilateral great auricular nerve,and in the xenogeneic acellular nerve graft group,this was done using a xenogeneic acellular nerve graft with trunk-branches.Examinations of facial symmetry,nerve-muscle electrophysiology,retrograde transport of labeled neuronal tracers,and morphology of the regenerated nerve and target muscle at 8 months postoperatively showed that the faces of the monkey appeared to be symmetrical in the static state and slightly asymmetrical during facial movement,and that they could actively close their eyelids completely.The degree of recovery from facial paralysis reached House-Brackmann grade II in both groups.Compound muscle action potentials were recorded and orbicularis oris muscles responded to electro-stimuli on the surgical side in each monkey.Fluoro Gold-labeled neurons could be detected in the facial nuclei on the injured side.Immunohistochemical staining showed abundant neurofilament-200-positive axons and soluble protein-100-positive Schwann cells in the regenerated nerves.A large number of mid-graft myelinated axons were observed via methylene blue staining and a transmission electron microscope.Taken together,our data indicate that xenogeneic acellular nerve grafts from minipigs are safe and effective for repairing whole facial nerve defects in rhesus monkeys,with an effect similar to that of autologous nerve transplantation.Thus,a xenogeneic acellular nerve graft may be a suitable choice for bridging a whole facial nerve defect if no other method is available.The study was approved by the Laboratory Animal Management Committee and the Ethics Review Committee of the Affiliated Wuxi No.2 People's Hospital of Nanjing Medical University,China(approval No.2018-D-1)on March 15,2018.

Proximity association in polygynous western black crested gibbons(Nomascus concolor jingdongensis):network structure and seasonality

We investigated the structure and seasonality of the proximity network in a group of polygynous western black crested gibbons(Nomascus concolor)using social network analysis.The spatial proximity changed seasonally and was affected by temperature and rainfall.Preferred proximity association was not distributed randomly among individuals.Kinship was one explanation for the social structure,as offspring preferred to maintain close proximity with their mothers.The proximity of infants to mothers decreased with age,and independent offspring had lower proximity to mothers than dependent ones.We found that the adult male had different proximity relationships with two different adult females.The frequency of proximity between the male and the infant-carrying female was significantly higher than that between the male and the female who had immigrated carrying one offspring of uncertain paternity into the group.Infanticide avoidance and/or predation protection for dependent infants might explain the proximity relationship differences.Temperature influenced group proximity association,with individual proximity increasing in the cold months and decreasing in the hot months.Group proximity decreased in months with higher anthropogenic disturbance.

Two new SINE insertion polymorphisms in pig Vertnin(VRTN)gene revealed by comparative genomic alignment

Despite one SINE retrotransposon insertion polymorphism(sRTIP)in the vertebrae development-associated(VRTN)gene was identified in pigs,the structural variations(SVs)in VRTN gene and its proximal flank regions were largely unknown.VRTN genic and flanking sequences from 14 breeds were assembled or downloaded from whole genome shotgun contings(WGS)database,and aligned to identify the SVs with Clustalx,and retrotransposons in VRTN gene were annotated by RepeatMasker,the splicing patterns of VRTN gene were predicted by Genescan,and large SVs were evaluated by PCR.A total of 12 small SVs and three large SVs in intron of VRTN,derived from SINE insertion polymorphisms.were identifed,and two of them(VRTN-sRTIP2 and VRTN-sRTIP3)were not reported before.These VRTN-sRTIPs may affect the splicing patterns of VRTN.They displayed polymorphisms in most detected eight breeds.VRTN-sRTIP2 and VRTN-sRTIP3 showed Hardy-Weinberg equilibrium distributions in most populations except the Chinese local Erhualian pigs,while VRTN-sRTIP1 showed genetic equilbrium in Erhualian pigs.Three VRTN-sRTIPs were identified,and displayed polymorphisms in pigs,and two of them were not reported before.These SVs provide a useful molecular markers for genetic analysis in pigs,and offer new information to facilitate the understanding the SVs of VRTN gene and their putative roles in the variation of vertebral number.

A BAC clone of MDV strain GX0101 with REV-LTR integration retained its pathogenicity

The complete genome of Marek's disease virus (MDV) strain GX0101, which was integrated with the LTR sequences of REV, was cloned in Escherichia coli as a bacterial artificial chromosome (BAC). BAC vector sequences were introduced into the US2 locus of the MDV genome by homologous recombination. The viral DNA containing the BAC vector was used to transform Escherichia coli strain of DH10B. Then the recombinant virus was successfully rescued by transfection of the recombinant BAC DNA into primary chicken embryo fibroblast (CEF). This BAC viral clone was named bac-GX0101. When the reconstituted virus was inoculated into 1-day-old birds, visceral tumors could be detected as early as 62 d post infection. There was no difference in growth ability and pathogenicity to birds between the BAC derived virus and its parental virus. The BAC derived virus maintained its oncogenicity and immunosuppressive effects. In conclusion, the complete genome of GX0101 strain was successfully cloned into BAC and the infectious clone was rescued. With the powerful BAC manipulation system, the infectious clone will provide a useful tool for further understanding the functional roles of the inserted REV-LTR sequence in the GX0101 strain of MDV.

Establishment of a transgenic mouse model with liver-specific expression of secretory immunoglobulin D

Mutation of mevalonate kinase （MVK） is thought to account for most cases of hyperimmunoglobulinemia D syndrome （HIDS） with recurrent fever. However, its mechanism and the relationship between elevated serum immunoglobulin D （IgD） and the clinical features of HIDS are unclear. In this study, we generated by fusion PCR a vector to express high levels of chimeric secretory IgD （cslgD） specifically in the liver. We then generated seven founder lines of transgenic mice by co-microinjection, and verified them using genomic PCR and Southern blotting. We detected the expression of csIgD by reverse transcription PCR, quantitative PCR, western blotting, and enzyme-linked immunosorbent assays. We demonstrated that csIgD could be specifically and stably expressed in the liver. We used flow cytometry to show that overexpression of csIgD in the bone marrow and spleen cells had no effect on B cell development. Morphologic and anatomical observation of the transgenic mice revealed skin damage, hepatosplenomegaly, and nephromegaly in some transgenic mice; in these mice, pathological sections showed high levels of cell necrosis and protein-like sediments in the liver, spleen, and kidney. We demonstrated that the genomic insertion sites of the transgeues did not disrupt the MVK gene on mouse chromosome 5. This transgenic mouse will be useful to explore the pathogenesis of HIDS.

A Single-cell Transcriptome Atlas of Cashmere Goat Hair Follicle Morphogenesis

Cashmere,also known as soft gold,is produced from the secondary hair follicles(SHFs)of cashmere goats.The number of SHFs determines the yield and quality of cashmere;therefore,it is of interest to investigate the transcriptional profiles present during cashmere goat hair follicle development.However,mechanisms underlying this development process remain largely unexplored,and studies regarding hair follicle development mostly use a murine research model.In this study,to provide a comprehensive understanding of cellular heterogeneity and cell fate decisions,single-cell RNA sequencing was performed on 19,705 single cells of the dorsal skin from cashmere goat fetuses at induction(embryonic day 60;E60),organogenesis(E90),and cytodifferentiation(E120)stages.For the first time,unsupervised clustering analysis identified 16 cell clusters,and their corresponding cell types were also characterized.Based on lineage inference,a detailed molecular landscape was revealed along the dermal and epidermal cell lineage developmental pathways.Notably,our current data also confirmed the heterogeneity of dermal papillae from different hair follicle types,which was further validated by immunofluorescence analysis.The current study identifies different biomarkers during cashmere goat hair follicle development and has implications for cashmere goat breeding in the future.

Metabolic properties of chicken embryonic stem cells

Cellular energy metabolism correlates with cell fate,but the metabolic properties of chicken embryonic stem (chES) cells are poorly understood.Using a previously established chES cell model and electron microscopy (EM),we found that undifferentiated chES cells stored glycogen.Additionally,undifferentiated chES cells expressed lower levels of glucose transporter 1 (GLUT1) and phosphofructokinase (PFK) mRNAs but higher levels of hexokinase 1 (HK1) and glycogen synthase (GYS) mRNAs compared with control primary chicken embryonic fibroblast (CEF) cells,suggesting that chES cells direct glucose flux towards the glycogenic pathway.Moreover,we demonstrated that undifferentiated chES cells block gluconeogenic outflow and impede the accumulation of glucose-6-phosphate (G6P) from this pathway,as evidenced by the barely detectable levels of pyruvate carboxylase (PCX) and mitochondrial phosphoenolpyruvate carboxykinase (PCK2) mRNAs.Additionally,cell death occurred in undifferentiated chES cells as shown by Hoechst 33342 and propidium iodide (PI) double staining,but it could be rescued by exogenous G6P.However,we found that differentiated chES cells decreased the glycogen reserve through the use of PAS staining.Moreover,differentiated chES cells expressed higher levels of GLUT1,HK1 and PFK mRNAs,while the level of GYS mRNA remained similar in control CEF cells.These data indicate that undifferentiated chES cells continue to synthesize glycogen from glucose at the expense of G6P,while differentiated chES cells have a decreased glycogen reserve,which suggests that the amount of glycogen is indicative of the chES cell state.

Improvement in bioconversion efficiency and reduction of ammonia emission by introduction of fruit fermentation broth in a black soldier fly larvae and kitchen waste conversion system

The black soldier fly(BSF),Hermetia illucens(Diptera:Stratiomyidae),is an insect commonly used for the bioconversion of various organic wastes.Not only can the BSF convert organic waste into macromolecular organic substances,such as insect pro-teins,but it can also lessen the pollution associated with these waste products by reducing ammonia emissions,for example.In this study,we measured the effects of adding fruit fermentation broth(Fer)and commercial lactic acid bacteria fermentation broth(Em)to kitchen waste(KW),as deodorizing auxiliary substances,on the growth performance of black soldier fly larvae(BSFL),the intestinal flora structure of BSFL,the ammonia emis-sion from the KW substrate,and the microbial community structure of the KW substrate.We found that the addition of Fer or Em increased the body weight of BSFL after 6 d of culture,increasing the growth rate by 9.96%and 7.96%,respectively.The addition of Fer not only reduced the pH of the KW substrate but also increased the relative abundance of probiotics,such as Lactobacillus,Lysinibacillus,and Vagococcus,which inhibited the growth of ammonifiers such as Bacillus,Oligella,Paenalcaligenes,Paenibacillus,Pseu-dogracilibacillus,and Pseudomonas,resulting in the reduction of ammonia emission in the KW substrate.Moreover,the addition of Fer or Em significantly increased the rela-tive abundances of Bacteroides,Campylobacter,Dysgonomonas,Enterococcus,and Ig-natzschineria in the gut of BSFL and increased the species diversity and richness in the K W substrate.Our findings provide a novel way to improve the conversion rate of organic waste and reduce the environmental pollution caused by BSF.

Diets enriched with finely ground wheat bran alter digesta passage rate and composition of the gut microbiome in sows

We investigated the effects of finely ground wheat bran on the nutrient digestibility, digesta passage rate,and gut microbiota structure in sows. A 3 × 3 Latin square design with 3 test periods and 3 experimental diets was used. Six non-pregnant sows(parity: 5 to 7) were randomly assigned to 3 experimental diets with 2 replicates per treatment in each period. Each period lasted 19 d(12 d for adaptation and 7 d for experiment). The experimental diets included(a) a basal corn and soybean meal diet(CON),(b) a basal diet with 20% coarse wheat bran(CWB;particle size: 605 μm), and(c) a basal diet with 20% fine wheat bran(FWB;particle size: 438 μm). The results demonstrated that the apparent total tract digestibility of neutral detergent fiber, acid detergent fiber and energy were reduced(P < 0.05) in the FWB and CWB groups compared with those in the CON group. Viscosity of digesta increased(P < 0.001) in FWB-fed sows. The passage rate of digesta from the mouth to the ileum decreased(P < 0.001) in FWB-fed sows. Peptide YY(PYY) concentration increased(P = 0.01) in FWB-fed sows after 30 min of feeding.In the FWB group, the relative abundance of Lactobacillaceae at the family level increased(P < 0.05) in the ileal digesta. At the class level, the relative abundance of Clostridia in feces decreased(P < 0.05) in FWB-fed sows. FWB enhanced the concentration of butyrate in feces compared with CON and CWB(P = 0.04). These results suggest that dietary supplementation with finely ground wheat bran reduces the passage rate of digesta, increases the abundance of beneficial microorganisms, and elevates the concentration of short-chain fatty acids and PYY in sows. These findings indicate that the addition of finelyground wheat bran to the diets of sows is more effective than using coarse wheat bran for improving their satiety and intestinal microbial composition.