Crosstalk between adipose and muscular tissues is hypothesized to regulate the number of muscular and adipose cells during fetal growth, with post-natal consequences on lean and fat masses. Such crosstalk largely remains, however, to be described. We hypothesized that a characterization of the proteomes of adipose and muscular tissues from bovine fetuses may enhance the understanding of the crosstalk between these tissues through the prediction of their secretomes and surfaceomes. Proteomic experiments have identified 751 and 514 proteins in fetal adipose tissue and muscle. These are mainly involved in the regulation of cell proliferation or differentiation, but also in pathways such as apoptosis, Wnt signalling, or cytokine-mediated signalling. Of the identified proteins, 51 adipokines, 11 myokines, and 37 adipomyokines were predicted, together with 26 adipose and 13 muscular cell surface proteins. Analysis of protein–protein interactions suggested 13 links between secreted and cell surface proteins that may contribute to the adipose–muscular crosstalk. Of these, an interaction between the adipokine plasminogen and the muscular cell surface alpha-enolase may regulate the fetal myogenesis. The in silico secretome and surfaceome analyzed herein exemplify a powerful strategy to enhance the elucidation of the crosstalk between cell types or tissues. Semitendinosus muscle from Charolais fetuses allowed a mapping of 296 proteins with abundances modified across the fetal life [An adequate development of adipose tissue (AT) relative to muscle  is central both for issues related to the human health and to the meat industry. Indeed, excessive fat accumulation is related to human obesity and related pathologies, such as metabolic syndrome, insulin resistance, or cardiovascular diseases. Moreover, fat and muscle masses within the bodies of species of agronomic interest are among the major determinants of carcass economic value in the meat industry ,2. The ltal life ,8,9. Higtal life ,8,9. Thetal life ,8,9. In tal life ,11. The tal life  was limital life , and remThe main objective of the present study was to characterize fetal adipose and muscular proteomes in order to identify proteins that may sustain an “adipose–muscular” crosstalk, and thus contribute to the regulation of the balance between muscle and AT growth. The fetal age of 140 days post-conception (dpc) was chosen because both adipose and muscular tissues grow by hyperplasia and hypertrophy . Moreovep < 0.05 one-factor ANOVA) abundance between the two AT), while 514 proteins were identified in Semitendinosus and Longissimus thoracis muscles . Of these, 704 and 490 bovine proteins from AT and muscles were assigned to human orthologs (p < 0.01) GO terms related to biological processes were found for both muscle  and AT (373 human orthologs) were annotated by GO terms related to specialized functions of muscular and adipose tissues. Within the muscle dataset, several GO terms were grouped in the functional category “muscle development and functioning”, which annotated 45 proteins  with GO In order to identify the most complete secretome, computational predictions were realized using the bovine sequences of proteins identified in the present study, supplemented by those previously identified in bovine adipose tissue  and muscBecause some proteins are secreted through cellular pathways that do not involve a signal peptide, ProteINSIDE predicted proteins that may be secreted either by endosomal recycling, plasma membrane transporter, membrane flip-flop, or membrane blebbing, which involves formation of vesicles or exosomes. As the merged results of the 3 methods of prediction , ProteINSIDE proposed 10 and 20 additional proteins potentially secreted by the muscle and the AT, respectively, with 8 of these proteins being secreted by both tissues . Thus, 4The sequences of 750 adipose and 531 muscular proteins were also used for surfaceome prediction. From the bovine protein sequences, the BUSCA web server predicted 7 adipose and 4 muscular proteins present at the cell surface, 2 of which were (PSMC1 and F3) identified in both tissues . When geIn order to understand the “adipose–muscular” crosstalk, known protein–protein interactions (PPI) between proteins secreted by one tissue and proteins present at the cell surface of the other tissue were searched. Regarding proteins secreted by AT that may interact with muscular cell surface proteins, 2 PPI between 4 distinct proteins were identified A. The idRegarding proteins secreted by muscles that may interact with cell surface proteins of the adipose cells, 11 PPI between 16 proteins were identified B. The seSemitendinosus and Longissimus thoracis muscles as well as perirenal and omental ATs for the same fetuses. It provides an accurate description of the main functional pathways and biological functions involved in myogenesis, adipogenesis, as well as the first description of the secretome and surfaceome as a prerequisite to understand the crosstalk between fetal adipose and muscular tissues. The most novel results concern the prediction of secreted and cell surface proteins. Indeed, 51 putative adipokines, 11 myokines, 37 adipomyokines, and 26 adipose and 13 muscular cell surface proteins were predicted, and thus may contribute to the crosstalk between AT and muscles during bovine fetal growth.This study is the first to establish an enriched proteome of Semitendinosus muscle [Regarding previously published proteomes of fetal perirenal AT  or Semits muscle ,8, the ds muscle , a mastes muscle , and thes muscle . Interess muscle  and CCAAs muscle . In bovis muscle ,26), esps muscle ,28), alos muscle ). These s muscle . Basicals muscle ,30. At 1Longissimus dorsi muscle [Semitendinosus muscle [Of the new pathways identified by GO analysis that may contribute to the bovine adipogenesis and myogenesis, the present study has identified proteins related to apoptosis, Wnt signalling, and cytokine-mediated pathways. A large number of GO terms were related to apoptosis, mainly to the negative regulation of apoptosis. These data are in agreement with the results from bovine adult i muscle , as wells muscle , revealis muscle . Among ts muscle  and colls muscle  in L6 mys muscle , howevers muscle . Of the s muscle , while Ts muscle . HSPA5 , regulas muscle , KNG1 [5s muscle , and LGAs muscle ), or muss muscle ). Our sts muscle ), lipid s muscle ), autophs muscle  and RAB1s muscle ), or cels muscle ). From ts muscle , but alss muscle , CAP2 [6s muscle , CTSD [6s muscle , and PSMs muscle  were shos muscle  was shows muscle  was shows muscle , CAPNS1 s muscle , RPSA [2s muscle , and MSNs muscle  were rels muscle  and CTSBs muscle  were shos muscle  have bees muscle  was shows muscle , or Ras-s muscle  was showThese putative secreted or cell surface proteins may also contribute to the “adipose–muscular” crosstalk through interaction. Among the proteins potentially secreted by AT and having a potential role in the regulation of muscle growth, we highlight an interaction between the adipokine plasminogen (PLG) and the muscular cell surface protein Alpha-enolase (ENO1). ENO1 is mainly described for its role as an intracellular glycolytic enzyme, however its presence as a cell surface protein has been already reported in muscular cells, particularly on the cell surface of differentiating myocytes . High exSemitendinosus (ST), and Longissimus thoracis (LT) muscles were sampled. Tissues were stored at −80 °C pending protein extraction. All procedures involving animals were approved by the North Dakota State University (NDSU) Animal Care and Use Committee (#A10001).Fifteen fetuses were generated by artificial insemination of multiparous crossbred beef cows (predominately of Angus breeding) at the North Dakota State University, Beef Research and Teaching Unit , as previously described . FetusesSemitendinosus and 15 from Longissimus thoracis muscles) were matched to a reference gel chosen from the 30 gels from one tissue type. We detected 368 muscular and 359 adipose well-resolved protein spots. The maximum number of protein spots was sampled for identification.Total proteins were extracted as previously described from AT  and fromGels were washed with distilled water and protein spots were excised individually and placed in sterile Eppendorf tubes. Spots were discolored in 100 µL of 25 mM NH4HCO 3–5% acetonitrile buffer over 30 min, and then in 100 µL of 25 mM NH4HCO 3–50% acetonitrile buffer for 30 min twice. Spots were then dehydrated for ten minutes in 200 µL of 100% acetonitrile. Acetonitrile was removed and samples were dried during 10 min using a Speed Vac . Proteins were hydrolyzed in peptides using 150 ng of trypsin solution (12 µL of a 25mM NH4HCO3 containing 12.5 ng/µL of typsin). After 5 h of incubation at 37 °C, peptides were extracted using sonication with 9.6 µL of 100% acetonitrile. Supernatants were transferred in High-Performance Liquid Chromatography (HPLC) vials and dried using a Speed Vac for 10 min. Then, 10 µL of equilibration solution (H2O/formic acid-100/0.5) was added before Nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis.m/z 400–1400), dynamic exclusion used with 2 repeat counts, 30 s repeat duration, and 60 s exclusion duration. For MS/MS, the isolation width for the ion precursor was fixed at 2 m/z and single charged species were rejected; fragmentation used 37% normalized collision energy as the default activation of 0.25.The peptides mixtures were analyzed by nano-LC-MS/MS using an Ultimate 3000 system  coupled to an LTQ velos  mass spectrometer with a nanospray ion source. After injection, 8 µL of peptide mixture was concentrated at a flow rate of 30 µL/min with a trap column  equilibrated with trifluoroacetic acid 0.05% in water. After 6 min of desalting, a valve was switched and the separation of peptides mixture was carried out according to their hydrophobicity with a nanocolumn  equilibrated with 0.5% formic acid in water. The solvent gradient increased linearly from 4% to 90% acetonitrile in 0.5% formic acid at a flow rate of 300 nL/mn for 38 mn. The peptides eluate was electrosprayed in positive-ion mode at 1.6 kV through a nanoelectrospray ion source operated in Collision-induced dissociation top 5 mode . The parameters of mass spectrometry analysis were as follows: full-enhanced-scan MS spectra acquired with 1 microscan . The database used for the search was UniProt Bos taurus , with the following parameters: peptide mass tolerance was set to 1.5 Da, fragment mass tolerance was set to 0.5 Da, and a maximum of two missed cleavages were allowed. Variable modifications were methionine oxidation (M) and carbamidomethylation (C) of cysteine. In this analysis, a peptide is validated when its Mascot score is greater than 39, which indicates a significant match for the announced sequence  < 5%). Protein identification was considered valid if at least 3 peptides with a statistically significant Mascot score were assigned it. Criteria of FDR < 5% rather than < 1% plus 3 peptides were chosen to maximize the discovery of adipose and muscular proteins for a large description of the secretome and surfaceome. Proteomics analyses were carried out on the two anatomical sites of adipose tissue or muscle in order “maximize” the discovery of adipose or muscular proteins for the most complete description of the secretome, surfaceome, and the crosstalk between tissues.At the end of each LC-MS/MS analysis, the raw files were loaded into the Proteome Discoverer v1.4  and identification was performed with Mascot v2.5 (https://www.proteinside.org). The UniProt identifiers of the bovine proteins identified in adipose and muscular tissues were automatically translated into human identifiers using orthologous and homologous links that are known between the two species and proposed by ProteINSIDE to take advantage of the most complete annotation available for human genes. Indeed, as genes linked by evolution, particularly orthologs, usually have conserved functions between vertebrates and because the human is the most annotated species, human orthologs of bovine proteins were considered in order to further investigate their biological functions. Either human or bovine identifiers were, used depending on the step of the data mining workflow  in order to identify biological pathways associated with fetal adipogenesis and myogenesis. The GO enrichment analyses were achieved in the human species using human orthologs. The Benjamini–Hochberg (BH) adjusted p-values were considered to establish lists of significant enriched pathways in each dataset as compared to the whole genome. Only GO terms with BH p-values < 0.01 that had annotated at least 2 proteins were considered to describe the adipose and muscular fetal datasets. Additionally, biological functions related to adipogenesis and myogenesis were searched from results of the IDresume module provided by ProteINSIDE, gathering descriptions of protein functions from Uniprot and NCBI databases.Identifiers for adipose and muscular proteins were launched independently for a basic GO analysis  through a local version of the SignalP algorithm . The prehttp://busca.biocomp.unibo.it/; Bologna Biocomputing Group, Bologna, Italy) [The surfaceome was defined as all plasma membrane proteins that have at least one amino acid residue exposed to the extracellular space . The exp, Italy) . The BUSWe applied two strategies to predict the bovine fetal adipose and muscular surfaceomes. The first one was the prediction of the surfaceome using the BUSCA web service for eukaryota and animals. For this prediction, we used the bovine protein sequences, with the following criteria used to declare a protein as putatively located at the cell surface membrane: the presence of at least one α- helical or β-barrel transmembrane domain or of a glycosylphosphatidylinositol (GPI) anchor plus the GO term annotation GO:0005886 for the plasma membrane. The second strategy aimed to compare the gene names of the 750 adipose and 531 muscular proteins to the gene names of the plasma membrane proteins identified by complementary predictive methods. Thus, we built a compendium of cell membrane proteins by merging 2882  and 3702https://www.uniprot.org) [https://www.ebi.ac.uk/intact/main.xhtml) [https://thebiogrid.org) [https://www.ebi.ac.uk/gxa/home) [Interactions between proteins secreted by a tissue and cell surface proteins from another tissue were searched with the Protein–Protein Interactions (PPI) module of ProteINSIDE. PPI between the proteins were identified thank to the PPI referenced within UniProt (rot.org) , IntAct n.xhtml) , and Biorid.org)  databaserid.org) , these Prid.org) ) or MIMIrid.org) ). Only Pxa/home) .To conclude, this study provides new data on the molecular events underpinning simultaneously bovine myogenesis and adipogenesis at a fetal age corresponding to increases in both cell number and volume for AT and muscle. These data are valuable for issues related to the lean-to-fat ratios of bovine carcasses and meat for the beef industry, but also for knowledge relevant to the ontogenesis of human body composition. Indeed, available data revealed that myogenesis in cattle is very close to that of humans . A large part of the data provided by the present study remains to be explored, and thus are made available for AT and muscle physiologists interested in understanding the fetal growth of these tissues.


Chicken manure is an agricultural residue material with a high biomass potential. The energetical utilization of this feedstock via anaerobic digestion is an interesting waste treatment option. One waste treatment technology most appropriate for the treatment of stackable (non-free-flowing) dry organic waste materials is the dry batch anaerobic digestion process. The aim of this study was to evaluate the substrate suitability of chicken manure from various sources as feedstock for percolation processes. Chicken manure samples from different housing forms were investigated for their chemical and physical material properties, such as feedstock composition, permeability under compaction and material compressibility. The permeability under compaction of chicken manure ranged from impermeable to sufficiently permeable depending on the type of chicken housing, manure age and bedding material used. Porous materials, such as straw and woodchips, were successfully tested as substrate additives with the ability to enhance material mixture properties to yield superior permeability and allow sufficient percolation. In dry anaerobic batch digestion trials at lab scale, the biogas generation of chicken manure with and without any structure material addition was investigated. Digestion trials were carried out without solid inoculum addition and secondary methanization of volatile components. The specific methane yield of dry chicken manure was measured and found to be 120 to 145 mL/g volatile solids (VS) and 70 to 75 mL/g fresh matter (FM), which represents approximately 70% of the methane potential based on fresh mass of common energy crops, such as corn silage. According to Haenel et al. [3 emissions of poultry breeding have a significant share (9%) of the overall NH3 emissions from animal husbandry. More than half of these emissions are caused by manure handlingDue to increasing protein demand for human nutrition, poultry farming is one of the fastest developing sectors of animal husbandry. The FAO stated that, over the last fifty years, poultry was the livestock category with the highest head growth worldwide, with roughly a five-fold increase . In Germl et al.  the NH3 3/t. The average methane yields of cattle dung, cattle manure and pig manure are comparably lower with 33–36 m3/t, 11–19 m3/t and 12–21 m3/t [Chicken manure is an agricultural waste material, which is a particularly interesting substrate for biogas production . Compare–21 m3/t  nitrogen–21 m3/t .The dry batch anaerobic digestion process is a waste treatment technology most appropriate for the treatment of stackable (non-free-flowing), fibrous and contaminant-containing dry feedstock materials. In contrast to conventional wet digestion systems, these systems rely purely on percolation of process fluid through the feedstock material within a garage type dry digester to facilitate the digestion process . ProcessThe advantage of this type of dry batch digestion process is a low susceptibility to contaminants, such as sand, stones, glass, plastics or metal, which can lead to an impairment of the stirrer and pump technology in a stirred tank system. Furthermore, the system is relatively unaffected by substances that tend to create sediment layers on the fermenter floor. Since the box system is opened, emptied and recharged after completion of a single fermentation cycle, larger sediment layers on the floor are avoided. In agricultural wet digestion systems large amounts of liquid are required i.e., liquid manure to adjust the water content and viscosity of feedstock mixtures with high dry matter contents. This results in the generation of high volumes of diluted digestate, which increases the costs of digestate storage and transport for agricultural use . In the The dry batch anaerobic digestion process requires a feedstock material with sufficient permeable structure that enables percolation of liquids in the anaerobic stage to ensure the mentioned transportation function ,14. InsuThe aim of this study was to examine the feedstock suitability of chicken manure for dry batch digestion with regards to material composition and permeability under compaction. Hence, the novelty of this study surrounds oedometer tests that were conducted as a method for material characterization that guided conditioning practices to create an appropriate substrate material structure for dry anaerobic batch digestion of chicken manure at full scale. Furthermore, dry batch anaerobic digestion trials without solid inoculum addition and a secondary methanization digester were carried out with mixtures of chicken manure and different structure material contents. The aim of the digestion trials was to determine the digestibility of chicken manure without inoculation, and to evaluate the influence of structure material addition and material permeability on the specific methane yield. The ultimate goal is to increase the efficiency of percolation processes so that, on the one hand, new plants can enter directly into efficient operation (without long running-in times), and on the other hand, existing plants can optimize their operations and substrate utilization.Chicken manure samples were tested for their material properties including total solids and volatile solids, nitrogen, protein, fat and fiber composition, permeability under compression and compressibility. Additionally, dry anaerobic batch digestion trials were carried out as a means to evaluate the digestion properties of chicken manure mixtures without inoculum addition and a secondary methanization digester.Material characterization tests of chicken manure were carried out with samples collected during 2014 and 2020 from different regions of Saxonia in Germany. Wet sample materials were quickly cooled after sampling and stored at 5 °C before lab analysis. Structure materials such as beech wood chips, wheat straw and plastic carrier  were provided by the Deutsches Biomasseforschungszentrum gemeinnützige GmbH (DBFZ).4-N) and total ammonia nitrogen (TAN) were determined as described in Liebetrau et al., (2016) [FA) concentrations were calculated according to Equation (1)NH4 is the total ammonium concentration, pH is the pH-value and t stands for temperature.A multitude of material characterization tests were performed on chicken manure samples and substrate mixtures of chicken manure after structure material addition. Total solids (TS) and volatile solids (VS) were measured in accordance with DIN EN 12880 (2001)  and DIN , (2016) . Free amIn order to determine the influence of the material compaction on substrate permeability and compaction, oedometer tests were conducted to simulate full scale conditions inside a percolation digester. A schematic illustration of the testing stand can be seen in Q (m3/s) through the sample, which is calculated from the volume of water Vw (L) passing through the sample material per unit of time t (s).The sample material was placed in an oedometer with a 254 mm diameter and 240 mm height and compressive force was applied with a perforated compression plate with 247 mm diameter. The compression plate was connected to an air piston as seen in fk (m/s) describes the ease with which the percolate can move through the pore spaces of the sample material. It is calculated by the quotient of the flow rate Q (m3/s) multiplied by the material sample height l (m) divided by the material surface area in the oedometer A (m2) multiplied by the hydrostatic height h (m).The hydraulic conductivity or permeability Sh (m) is calculated with the defined force applied by the perforated plate connected to the air piston and the oedometer diameter d (m), the density of the water saturated sample ρ (kg/m3) and the mass m (kg) of the sample material describe the simulated material volume. The cumulative compaction or material compressibility is determined by dividing the material height before and after compaction.The simulated material heap height P1) and with 1.5 m (P2) and 3.0 m (P3) simulated feedstock heap height. The material compressibility (C2) and (C3) was measured at the two mentioned compression levels.The test setup was configured to measure the material permeability without compaction  of the substrate mixture to a heap height of app. 0.3 m.Dry batch anaerobic digestion—in three test series, defined dry chicken manure and structure material mixtures were fermented in two percolate digesters. Each of the individual fermentation experiments were completed after a test period of approximately four weeks. Information about the type of structure material used and the percentage of its addition to dry chicken manure can be found in 4. The volatile fatty acid concentration in the percolate was measured on a daily base in the first week of the test and 2 to 3 times per week after the first week.The testing system consisted of two percolate digesters, each connected to a percolate pump for percolate recycling. The two double walled percolate digesters each had a substrate capacity of app. 13 L and were heated to 37 °C by a thermostatically controlled water bath. The hydrolysis and methanization occurred simultaneously in the percolate digesters. Each digester was filled with 2 kg of chicken manure with and without structure material addition. The material mixture compositions that were used are shown in Pearson correlation analysis was performed to determine the strength of the relationship between physio-chemical characteristics  and methane production after 35 days batch duration. Pearson’s correlation coefficients were performed using SAS software .The umbrella term chicken manure can be differentiated and includes chicken slurry, dry chicken excreta, dried chicken excreta, chicken manure, fresh chicken excreta. The chicken manure types can differ in the origin or bird purpose and by the chicken housing style i.e., broiler, laying hens, young animals. Chicken manure is the mixture of feces and urine excreted, which contains varying amounts of undigested feeding stuff, desquamated intestinal epithelium, residues of secretion, microorganism from the intestinal flora, metabolites excreted with the urine, as well as other components e.g., feather, egg leftovers, bedding material, grid material and soil. In this study, chicken manure was tested for the material properties of total solids (TS) and volatile solids (VS), nitrogen, protein, fat and fiber composition. The 42 tested samples were derived from various sources and varied in storage age, housing form and bedding material type. The material characterization tests were carried out with the aim of obtaining an overview of the material properties and material diversity. The results are summarized in 4-N) content was approx. 50% of total nitrogen. The calculated raw protein content of the 42 tested samples varied between 21.6% and 46.2%TS. The material characterization results confirm the findings of Nie et.al., 2015 [The Total Kjeldahl Nitrogen (TKN) content of 42 tested chicken manure samples from various sources was in a range of approx. 1.5 to 8.7%TS. On average the ammonium  showed similar permeability properties for all tested chicken manure samples  concentrations ranged from 1.6 to a maximum of 7.2 g/L (4+) and free ammonia (NH3) are the predominant forms of inorganic nitrogen. Ammonium nitrogen (NH4-N) is a degradation product of organic nitrogen such as proteins or urea during anaerobic digestion. In the digester liquid, NH4-N is present as ammonium ions (NH4+) and as free ammonia (NH3). Increasing pH or temperature, results in a higher percentage of NH4-N present as free ammonia (NH3). It has been shown that free ammonia is more toxic, as it can pass through the cell membrane, causing proton imbalance and potassium deficiency [4-N concentration range between 1.5 to 5.0 g/L. However, significantly higher concentrations of up to 14 g/L are possible by microbial adaptation of the digestion process. Niu et al. [The pH varied between 6.8 to 8.2 during the course of the digestion experiments a. During 7.2 g/L c and fre 7.2 g/L d. Accordficiency . Accordificiency , Bujoczeficiency  and Yenificiency , severalu et al.  describeIn our study, chicken manure achieved an average SMY of 135 ± 20 mL/g VS or 75 ± 5 mL/g FM. According to FNR , the aveThe methane concentration increased during the test duration to a maximum of approx. 68% as shown in In our study, chicken manure without structure material addition achieved SMYs of 127 ± 12 mL/g VS or 70 ± 6 mL/g FM . The FigThe SMY of the straw variants was 6% higher compared to the control variant without structure material addition and resulted in 135 ± 4 mL/g VS or 75 ± 2 mL/g FM. Methane production occurred without a lag phase in the beginning. The methane concentration of the biogas exceeded 50% after a test duration of approx. 8 days. Data to the gas composition is summarized in −5 m/s and achieved the highest specific methane yield. The detailed results of results of Pearson’s correlation coefficients are presented in The digestion of the wood chip variants resulted in the highest SMY with 143 ± 45 mL/g VS or 79 ± 25 mL/g FM. Although the average methane generation was 11% higher compared to the control without structure material addition, the high standard deviation between the two digesters has to be considered. Until day 20, the methane production rate was similar to the control variant. Between day 21 and 35, the methane production increased but varied in the parallel digesters see c. The prIt must be checked whether a very high use of structural substances is possible in full scale plant operation, because this reduces the plant throughput of the actual feedstock. Only very cost-effective structural materials, such as residual straw, were considered, since alternative structural materials, such as wood chips, are highly unlikely to be economically viable in practical application. A recovery of structural materials directly from the digestate is possible but challenging due to the unfavorable material properties that will impact the subsequent sieving. Under practical conditions, the use of screened residues as structure building material from chicken manure with bedding material appears to be more promising. In order to replace structural materials, which do not contribute to gas production in the mixture the material permeability of chicken manure can also be improved by mixing fibrous bedding material containing chicken manure with a high proportion of lignified biomass with moist, structurally weak chicken manure without bedding material, if both types of chicken manure are available simultaneously. A validation of the findings in full scale operations should be considered.Material characterization tests of chicken manure were carried out to determine the feedstock suitability in terms of feedstock composition, permeability under compaction and compressibility for dry batch anaerobic digestion processes. Oedometer tests under compression showed that the permeability and structural stability of chicken manure can be improved through an initial addition of structural materials such as wood chips or wheat straw to the substrate mixture. Even originally impermeable chicken manure samples were suitable for percolation processes after an addition of 5% straw or wood chips (by mass). Channel effects with uneven percolate distribution were observed on a number of samples with 10% woodchips addition. Anaerobic dry batch digestion trials were carried out to investigate the biogas generation of dry chicken manure without inoculation and separate methanization step. The conducted digestion trials showed that dry batch anaerobic chicken manure digestion was possible without inoculum addition. However, the methane production showed a lag phase of approximately 2 weeks after the test start, and the measured methane yields were lower compared to published SMYs for wet digestion systems. In this study the obtained methane yield of chicken manure averaged 135 ± 20 mL/g VS or 75 ± 5 mL/g FM, which represents 70% of the gas potential based on fresh mass of common energy crops such as corn silage. The addition of straw and wood chips led to an increase of the specific methane yield by 6% and 11%, respectively.


Membranes with interconnected pores with an average diameter of 60 µm and a contact angle of 97°, decorated with TiO2 nanoparticles, are obtained. The degradation of oily wastewater demonstrated the high photocatalytic efficiency of the nanocomposite membranes: Under sunlight irradiation for seven hours, colourless water was obtained.The compounds found in industrial wastewater typically show high toxicity, and in this way, they have become a primary environmental concern. Several techniques have been applied in industrial effluent remediation. In spite of the efforts, these techniques are yet to be ineffective to treat oily wastewater before it can be discharged safely to the environment. Membrane technology is an attractive approach to treat oily wastewater. This is dedicated to the immobilisation of TiO The production of oil and gas by petroleum refineries yields a large amount of wastewater, containing mostly organic compounds , inorganic compounds , and dissolved and suspended solids .Nowadays, many techniques are being employed in oily wastewater treatment, such as ultrafiltration , ultraso2)/PVDF-TrFE nanocomposite produced by the authors showed a remarkable solar photocatalytic activity in tartrazine degradation—degradation of 78% under five hours of sunlight irradiation [Furthermore, the hydrophobicity of polymeric membranes promotes their contamination in the filtration process, especially in oil/water separation, leading to the decrease of water flow and rejection efficiency . Membranadiation .2) is the most widely used photocatalyst for degradation of organic compounds due to its properties, such as non-toxicity, low cost and abundance, physical and chemical stability, superhydrophilicity, and superior photocatalytic activity under UV radiation (λ < 390 nm) [2 into polymeric membranes solves some drawbacks related to its use in suspension, namely the recuperation of the nanoparticles.As a catalyst, titanium dioxide (TiO 390 nm) . The incAn efficient alternative to applying photocatalytic membranes are the photocatalytic membrane reactors (PMRs). PMRs have shown a great potential as a “zero” waste process for oily wastewater treatment since they can reduce the loss of catalytic nanoparticles, control the contact time between catalyst and pollutant, and guarantee a continuous process. In this context, PMRs can improve the process efficiency and stability, and reusability of the nanocomposite to reduce the operating cost .2 nanoparticles immobilised on a PVDF–TrFE porous membrane as being an efficient method for the degradation of oil in wastewater in a solar photoreactor, making use of the UV radiation (3–5%) present in the solar radiation.Thus, the major focus of this work is to demonstrate TiO®-TiO2 nanoparticles were provided by EVONIK . Sulfuric acid (H2SO4) 99%, sodium hydroxide (NaOH) 97%, N,N-dimethylformamide (DMF) 99%, and methylene (MB) were all purchased from Merck .PVDF–TrFE (70:30) was obtained from Solvay . P252 nanoparticles [2 nanoparticles generously provided by Evonik  were added to 90 mL of N,N-dimethylformamide (DMF) obtained from Merck  and placed in an ultrasound bath for four hours to obtain a homogeneously dispersed solution. The amount of TiO2 used was previously chosen [The solvent casting technique was used to produce the PVDF-TrFE membranes with immobilised TiOarticles ,28. Brie2 nanoparticles was evaluated by X-ray diffraction (XRD) using a Bruker D8 Discover diffractometer with incident Cu Kα (40 kV and 30 mA). Fourier-transformed infrared spectroscopy (FTIR) was performed to determine the chemical stability of the nanocomposite using an FTIR Alpha instrument , over a range of 650–4000 cm−1 using 64 scans with a resolution of 4 cm−1. The scanning electron microscopy (SEM) was obtained with a Quanta 650 SEM  to access the morphology/microstructure of the membrane. Energy dispersive X-ray spectroscopy (EDX) was performed with an INCA 350 spectrometer , without gold coating.The crystal structure of the TiOThe wettability of the samples was measured through contact angle in Physics SCA20 microscope .The Hassi R’mel (HR) area is located in southern Algeria, 525 km south of Algiers, and 120 km south of Laghouat wilaya, about 70 km west of Beriane and 120 km south of Algiers, northwest of Ghardaia. The HR field is centered at 32° 55′41° North and 3° 16′16° East at an average elevation of approximately 750 m above sea level. The geometric shape, the nature of the effluent and the homogeneity of the reservoir at the HR field led to the selection of three production areas  and two compressor stations.The withdrawal of samples was accomplished on 15 March 2017, at the inlet of the de-oiling unit at CPI S-102 (Corrugated Plate Interceptor), which is a separating basin furnished with a set of parallel plates that facilitate the sedimentation of the non-decanted solid particles. The samples were transported from Hassi R’mel to the Research and Development Center in coolers and stored for 24 h in a refrigerator at 4 °C before being used.C0 and tC are the initial concentration of oxygen and concentration at time t, respectively.Several parameters were measured before and after the treatment. The temperature (T) was measured using a thermometer ASTM 5C and the electrical conductivity (EC) was measured by a conductivity meter HACH HQ 40d. The suspended solids (SS) were determined by filtering 100 mL of water to be analyzed through a 47 μm pore filter; afterwards, the filter was dried at 105 °C in an oven for two hours. The amount of suspended solids was estimated by the difference in the weight of the filter before and after drying. The pH measurements were carried out using a pH-meter type . Chemical oxygen demand (COD) was based on the oxidation of organic materials by an excess of potassium dichromate (K2Cr2O7), in acidic medium and boiling, in the presence of silver sulfate and mercury sulfate. COD was measured by a spectrophotometer of the type . The percentage of degradation of COD was estimated using the following Equation (1).The quantification of the total hydrocarbons (HC) in water followed an analytical protocol implemented according to the water standard methods .Total organic carbon (TOC) analysis was performed by TOC Analyzer Formacs HT. The determination of phosphate, nitrate, and nitrite was carried out using a DR1900 colourimetric spectrophotometer at a wavelength of 860 nm using the different LCK reagents.2/PVDF-TrFE nanocomposite membrane was placed at the bottom , using natural sunlight radiation. A Pyranometer CMP 11  with a spectral range between 285 and 2800 nm was used to measure the solar UV intensity. The reactor was developed at the Solar Equipment Development Unit (UDES) in Algeria. The capacity of the photoreactor is 1 L (38 cm length × 12 cm wide × 8.5 cm high). The photoreactor tank was fabricated from Pyrex glass, where the produced 8 wt.% TiOe bottom . The floFor the photocatalytic assays, one liter of oily wastewater solution was added to the photoreactor tank containing the membrane and exposed to solar illumination for seven hours, which is the period that assures a constant sunlight irradiance intensity during the experiment.The degradation of oily wastewater was analyzed with a UV–visible spectrophotometer , and the peak at 262 nm was used to monitor oily wastewater absorbance over the irradiation time.2 nanoparticles was assessed by X-ray diffraction (XRD) and compared with the pattern diffractograms  nanocomposite show a stable polymer structure concerning the pure polymer. Bands at 840, 1288 and 1400 cm−1 show that the polymer crystallizes in the all-trans piezoelectric β-phase [Infrared spectroscopy (FTIR) was used to determine the polymer phase and the possible chemical interaction between fillers and polymer matrix. In  β-phase .2 is present in the membranes, as already reported in Martins et al. [The polymer phase remains unchanged when TiOs et al. . In this2/PVDF-TrFE samples, which present contact angles of 97° and 76°, respectively. These results show the decrease of the contact angle with the incorporation of TiO2 nanoparticles on the polymer matrix, which in turn, allows for a higher interaction between the nanocatalyst and the oily water.Contact angle measurements assessed the wettability of the membranes. Before the photocatalytic assays, controls were performed using oily wastewater without membrane (photolysis), and oily water with pristine PVDF-TrFE (adsorption), and in both cases the degradation was neglectable (≈1%)—see results as This experiment has indicated an increased removal efficiency after diluting the initial oily water sample, with removal rates (R%) of 49% and 99.6% for the COD values of 51,758 and 25,879 mg/L, respectively. Such enhanced efficiency is related to turbidity reduction after dilution, from 425 NTU to 205 NTU. Decreasing turbidity favors the penetration of the sunlight radiation, which leads to oil removal rate improvement.2/P(VDF–TrFE) membranes were used to assess the degradation of oily wastewater in a solar photoreactor. The 8 wt.% TiOThe visual inspection indicates that the oily wastewater pollutants were degraded as colorless water was obtained after the seven hours. As confirmation, 2SO4) or sodium hydroxide (NaOH) solutions.In a different set of assays, the pH influence on the oily water degradation process was assessed; thus, three oily water solutions were prepared, and the initial pH was adjusted either with sulfuric acid  [The results obtained after seven hours of sunlight irradiation of the 8 wt.% TiOameters) . The tot2/PVDF-TrFE was formerly proved in [The reusability of the TiOroved in , which aThe disappearance of peaks in the chromatogram obtained by gas chromatographic analysis (GC-FID) confirms the efficient removal of total hydrocarbons, The majority of the organic compounds present in the sample before treatment a belong 2/PVDF-TrFE nanocomposite membrane, it is possible to treat oily industrial wastewater, such as that from the central region of Hassi Rmel Algeria, using the sun as a source of energy.The results show that by using the 8 wt.% TiO2/P(VDF–TrFE) nanocomposite by solvent casting and its photocatalytic application in the remediation of oily wastewater. The characterization of the samples shows a homogeneous distribution of the TiO2 nanoparticles over the pores with an interconnected structure. FTIR and contact angle results prove the chemical stability and the unchanged hydrophobic nature of the nanocomposite membrane. The photocatalytic assays allowed complete removal of the COD (99.6%), total hydrocarbons (99.6%), and phosphates (100%), as well as a significant reduction of the suspended solids (76%), turbidity (97%), TOC (98%), nitrates (69%), and chloride (26%) from oily wastewater. The pH range that allows better removal efficiencies ranges between 4 and 5.5. After seven hours of sunlight exposure, colourless oily wastewater was obtained. Thus, the fabricated membranes combine an inexpensive nanocatalyst and UV radiation source (available in sunlight radiation) to efficiently remediate oily water. Therefore, the TiO2/P(VDF–TrFE) nanocomposite membranes proved to possess a proper morphology and chemical stability envisaging oily wastewater remediation and related applications.This work reports the preparation of a TiO


At present, the posterior cervical approach with open reduction and internal fixation (ORIF) remains a commonly effective treatment for unstable Atlas fracture. However, the inserted screws into the C1 lateral mass of some unstable atlas fracture are very difficult, so that the operation is forced to change into C0 to C2 fusion. In order to improve the successful rate of lateral mass screw placement, we introduced a method of fixing lateral mass with a towel clamp in posterior transpedicular fixation, and explore the efficacy and feasibility.Twenty-one consecutive patients with unstable atlas fracture were treated via this method from October 2012 to July 2017. All cases had neck pain and restricted motion of neck movement on admission. Electronic medical records and pre- and postoperative radiographs were reviewed. Screw and rod placement, bone fusion, and spinal cord integrity were assessed via long-term follow-up with anteroposterior and lateral radiographs and computed tomography. Follow-up included clinical assessment of neurological function, assessment of pain using the visual analog scale (VAS), and assessment of the activities of daily living using the neck disability index (NDI).The mean follow-up duration was 22.1 months (range: 12–54 months). No screw loosening or breakage, plate displacement, neurovascular injury, and severe complications occurred during follow-up. The mean operative time was 112.4 ± 14.9 min (range: 82–135 min), and mean blood loss was 386.2 ± 147.9 mL (range: 210–850 mL). One patient experienced continuous neck pain postoperatively, but this gradually disappeared with analgesic administration. At final follow-up, all patients had bone fusion, the VAS scores and NDI were significantly improved compared with preoperatively.Fixing the C1 lateral mass with a towel clamp during posterior transpedicular fixation for unstable atlas fracture appears to be a safe and reliable method, with the advantages of being a simple technique with few complications. Unstable atlas fracture comprises an atlas burst fracture with simultaneous injury to the transverse atlantal ligament (TAL), characterized by outward displacement of the lateral masses, and atlantoaxial dislocation or subluxation. Unstable atlas fracture is very dangerous, as the displacement of bone blocks may cause spinal cord injury, leading to severe complications such as paraplegia and death. Hence, the stability of the atlantoaxial complex must be surgically reconstructed. Treatment aims to correct the dislocation, restore the stability of the atlantoaxial joint, and retain the maximum degree of motion of the cervical spine.Atlas fracture is rare, accounting for 3% to 13% of all cervical spine fractures, and 1.3% to 2% of all spinal injuries. Studies have described types of surgery performed for unstable atlas fractures, such as transoral approach anterior C1-ring plate osteosynthesis, posterior osteosynthesis with a lateral mass screw rod, and posterior C1 to C2 fusion and C0 to C2 fusion. However, anterior C1-ring plate osteosynthesis is prone to postoperative infection, and posterior osteosynthesis with a lateral mass screw rod can cause incomplete reduction of C1 anterior arch fractures; hence, these surgical methods are not widely used.,4 Although posterior fusion eliminates the mobility of the cervical spine, it is still useful for some types of unstable atlas fracture.The treatment of unstable atlas fracture remains controversial, and there is a lack of uniform standards or guidelines.,6 However, it is very difficult to fix the atlantoaxial joint in some unstable atlas fractures, as the unstable lateral mass swings or moves forward during the insertion of screws into the C1 lateral mass. Therefore, it may be necessary to perform C0 to C2 fusion instead of C1 to C2 fusion, which may cause other complications such as loss of activity of the atlantooccipital joint. To increase the success rate of screw insertion into the C1 lateral mass in unstable atlas fractures, we introduced a method in which a towel clamp is used to fix the lateral mass while the pedicle screw is inserted into the lateral mass. We consider that this method makes surgery easier and safer for displaced unstable atlas fractures. The present study aimed at to evaluate the safety and effectiveness of this novel method.Posterior C1 to C2 fusion with transpedicular fixation has the advantages of reliable fixation, few complications, and high fusion rates, while retaining part of the rotation and flexion extension; therefore, it is the most commonly used method for unstable atlas fracture.22.1This retrospective study was approved by the ethical review committee of our institution. Twenty-one consecutive patients  were diagnosed with unstable atlas fractures and treated surgically in our institution between October 2012 to December 2017 (Flow diagram). Inclusion criteria were1.inpatient due to trauma,2.suffered from C1 fracture (type III and IV),3.treated with the fixed lateral mass technique before inserting the screw,4.availability of radiographic examinations  and clinical data .Exclusion criteria were1.suffered from C1 fracture ,2.no surgery or incomplete date,3.associated with occipital condyle and odontoid fracture,4.other comorbidities, such as cervical stenosis, neoplasia, and infection.All cases were treated with posterior transpedicular fixation; a towel clamp was used to fix the lateral mass, and the pedicle screw was then inserted into the lateral mass. Unstable atlas fractures associated with occipital condyle and odontoid fractures were excluded. The electronic medical records and pre- and postoperative radiographs of each patient were reviewed. Mean patient age was 56.5 years (range 22–71 years). The causes of atlas fracture were: injury during a fall (n = 12), motor vehicle accident (n = 8), and injury by falling objects (n = 1). All patients had neck pain and stiffness with or without pain in other parts. Except for one patient in a transient coma, the rest were awake, cooperative, and had no neurological deficits at presentation. One patient also had C4/C5 traumatic disc herniation, while another had T4 burst fracture  version 19. Data were expressed as mean ± standard deviations for variables. Preoperative, post-operative 1 week and the final follow-up differences were performed using a paired 3P < .05) , and mean blood loss was 386.2 ± 147.9 mL (range: 210–850 mL) ; type II ; type III , which was further divided into type IIIa (intact TAL), and type IIIb (ruptured TAL); type IV ; and type V (isolated fractures of the C1 transverse process).There are several classification systems for atlas fractures, but none is uniformly accepted. The Jefferson, Landells, and Gehweiler classifications are most commonly used in clinical studies. The “rule of Spence” suggests that the TAL is probably torn in burst atlas fracture with displacement of the lateral masses of more than 6.9 mm; however, displacement of the lateral mass of <6.9 mm or without displacement of the TAL is likely to be overlooked, as plain radiography and CT do not provide direct visualization of the TAL.,17 The integrity of the TAL can be evaluated with high resolution magnetic resonance imaging. This is important in distinguishing between stable and unstable burst fractures.The TAL plays a crucial role in atlantoaxial stability, with atlas fractures often defined as stable or unstable based on the inferred integrity of the TAL. Dickman defined an intraligamentous rupture as Dickman type I, and bony avulsion as Dickman type II. It is widely accepted that surgery is indicated for atlas fracture associated with atlantooccipital instability, intraligamentous rupture of the TAL, and for “unstable” atlas fracture.,20 At present, there are several surgical methods with osteosynthesis for the treatment of unstable fractures, such as transoral approach anterior C1-ring plate osteosynthesis, posterior osteosynthesis with a lateral mass screw rod, and posterior C1 to C2 fusion and C0 to C2 fusion.–23 However, any surgical method has its advantages and disadvantages. For example, transoral approach anterior C1-ring plate osteosynthesis can retain important joint motion, but it can only repair of anterior 1/2 Jefferson fractures and has a high risk of wound infection; posterior osteosynthesis with a lateral mass screw rod can preserve rotary motion and prone to lower postoperative infection, but it is unable to stabilize fractured anterior arch and cause incomplete reduction of C1 anterior arch fractures; C0 to C2 fusion seems relatively stable, but its internal fixation shear force is larger, and it is prone to complications such as screw loosening and internal fixation failure. However fortunately, C1 to C2 fusion absorbs the advantages of the three operations mentioned above. It has reliable reduction, preserves the activity of the atlanto-occipital joint and is suitable for various unstable Jefferson fractures with lower risk of would infection, is a satisfactory treatment for unstable Jefferson fractures. Recent reports indicate that definitive atlantoaxial fusion is the current treatment of choice for elderly patients with unstable Gehweiler type 3b atlas fracture, as elderly patients have reduced bony healing ability, and isolated atlas osteosynthesis is not recommended in type 3b fractures with severe dislocated bony avulsion of the TAL.,24 This report provides a reference for the treatment of unstable Jefferson fracture.At present, the treatment of C1 fractures remains controversial, and there are no internationally accepted treatment standards. For isolated atlas fractures, conservative treatment is the main method, but for unstable atlas fractures, surgical treatment is still the preferred method.In the posterior approach for unstable atlas fracture, it is necessary to place a screw in the C1 lateral mass, irrespective of whether the fracture is being treated via osteosynthesis or atlantoaxial fusion. For Gehweiler type III atlas fractures and obviously displaced lateral mass fractures, the lateral mass swings or moves forward during C1 screw placement; this increases the risk of injury to the spinal cord and vertebral artery, which are potentially fatal complications. We use a towel clamp to fix the lateral mass, then drill the hole and insert the screw. The fixed position of the towel clamp is located at the stump of the posterior arch, with one side adjacent to the fracture line, the other side close to the lateral mass screw placement, and the upper edge adjacent to the vertebral artery. This technique is very important to temporarily stabilize the lateral mass during screw placement. It improves the success rate of screw placement in the C1 lateral mass, and avoids the implementation of C0 to C2 fusion and complications such as loss of activity of the atlantooccipital joint after fusion. It also reduces the risk of damaging important organs such as the spinal cord and the vertebral artery. In addition, it makes the operation easier, resulting in less damage and less bleeding.In the present study, 21 patients with an unstable atlas fracture were treated with C1 to C2 pedicle screw fixation and fusion after C1 lateral mass fixation with a towel clamp. The postoperative VAS and NDI was significantly lower than preoperatively. At final follow-up, three-dimensional CT showed satisfactory C1 to C2 bone fusion, with no broken screws, broken rods, and/or lateral mass separation. However, one patient had postoperative neck pain that was resolved after analgesia and neurotrophic treatment. There were two possible reasons for this pain. One is that in cases involving excessive traumatic force, atlantoaxial instability leads to cervical muscle spasm and severe edema after muscle and ligament damage, which stimulates the occipital nerve to cause pain; another is that there was no subperiosteal dissection during the surgical incision of the muscle, which may damage the occipital nerve or branch.The primary limitation of the present study is the small sample size. As this method is used in more cases, its safety and efficacy may be more thoroughly evaluated. Another limitation is the retrospective design; future prospective studies may better control for follow-up timing intervals, and could include more standardized outcome measures.5In conclusion, posterior transpedicular fixation while the lateral mass is fixed with a towel clamp is safe and reliable during treatment of unstable atlas fracture. This method is simple, enables effective fracture reduction, high fusion rates, and has few complications.Conceptualization: Jingwen Huang, Zengming Xiao.Data curation: Zhou Ding.Formal analysis: Wei Guo, Feng Hu.Funding acquisition: Zengming Xiao.Investigation: Wei Guo, Zhou Ding.Methodology: Wei Guo, Yang Lin.Resources: Wei Guo, Zengming Xiao.Software: Wei Guo.Supervision: Jingwen Huang, Feng Hu, Zengming Xiao.Validation: Wei Guo, Zengming Xiao.Visualization: Zengming Xiao.Writing – original draft: Wei Guo, Yang Lin.Writing – review & editing: Zengming Xiao.


CD93 on chromosome 20; this gene is not strongly expressed in the brain, however. The other signal was for AL118508, a non-protein coding pseudogene, which completely lies within CD93’s genomic coordinates, thereby explaining its signal. Neither are obvious candidates for involvement in the brain processes that underlie emotion recognition and its developmental pathways.The genetic contribution to different aspects of empathy is now established, although the exact loci are unknown. We undertook a genome-wide association study of emotional empathy (EE) as measured by emotion recognition skills in 4,780 8-year old children from the ALSPAC cohort who were genotyped and imputed to Phase 1 version 3 of the 1000 Genomes Project. We failed to find any genome-wide significant signal in either our unstratified analysis or analysis stratified according to sex. A gene-based association analysis similarly failed to find any significant loci. In contrast, our transcriptome-wide association study (TWAS) with a whole blood reference panel identified two significant loci in the unstratified analysis, residualised for the effects of age, sex and IQ. One signal was for  These skills are crucial in navigating our social milieu, in terms of independent living, vocational success and wider interpersonal relationships. Empathy itself, referring to the ability to share an emotional experience with another person, has been extensively studied from a biological perspective3. Two dimensions of empathy are widely recognised, namely emotional and cognitive empathy3. The former essentially describes the process of emotional isomorphism with another person, i.e. the sharing of their emotional state. Consequently, it requires the decoding of that information from cues offered by others’ facial expressions, vocalisations, and gestures4. In contrast, cognitive empathy refers to the ability to understand more complex mental states, and is therefore more closely aligned with the concept of theory of mind (ToM) or mentalizing7. From an evolutionary perspective, although attachment and parental care are observed across species, empathy is only truly developed in mammals that possess self-awareness, demonstrable among primates but very few other animals. Phylogenetically, emotional empathy is older and less complex, with ToM also requiring the ability to perspective take8.Our capacity to negotiate the complexity of the social world represents the remarkable evolutionary development of a wide range of mechanisms for the processing of social information. Some of our most advanced skills, such as language and empathy, mediate our interaction with our social environment9. These same dimensions have also been mapped onto brain networks using functional neuroimaging10. Although a full taxonomy is far from clear, the amygdala and its prefrontal and superior temporal connections are known to play a crucial role in the processing of social information, thereby mediating elements of empathy10.Using a variety of experimental paradigms research is now beginning to unravel the brain’s mechanisms for empathy. For example, much progress has been made in delineating the underlying neuropsychological dimensions of both emotional and cognitive empathy using a variety of experimental paradigms11 calculated a heritability of 67%  = 0.79, n.s., AIC = −7.21) for cognitive empathy for 3-year old twin pairs using a false belief paradigm, but by 5 years shared environment became increasingly important12. Autism Spectrum Disorder (ASD), a developmental disorder in which an abnormality of social interaction is a core impairment, is also known to be principally genetic in aetiology, with more than 100 genes or genomic regions harbouring rare genetic variants implicated so far13. Additionally, there is emerging evidence for the role of more common variants, sometimes coupled with rare variants, consistent with the genetic landscape of other neurodevelopmental and complex medical disorders15.Twin and family studies have demonstrated that the different aspects of empathy are heritable, but increasingly influenced by shared environmental factors as a child grows. Specifically, Hughes and Cutting16. Heritability for cognitive empathy was estimated at 5.8% , with a female specific locus at 3p26.1 reaching genome-wide significance. The leading SNP and the other 21 SNPs in high linkage disequilibrium with it are near LRRN1, which is highly expressed in brain tissue17. In a separate study, Warrier and Baron-Cohen18 also investigated the genetics of cognitive empathy using data from the Avon Longitudinal Study of Parents and Children, a prospective birth cohort study . Among teenagers, no GWA significant signal was identified, and SNP-based heritability was negligible . One limitation may have been the task used to measure cognitive empathy, the ‘Triangles Task’, which, although validated for ToM19, has little ecological validity.Recently, efforts have also been underway to identify common genetic variants that may be associated with different aspects of social cognition. For example, Warrier and colleagues conducted a genome-wide association (GWA) study of cognitive empathy using customers from 23 and Me and subjects from the Brisbane Longitudinal Twin Study20 investigated common variants associated with emotion recognition as measured by the Diagnostic Analysis of Nonverbal Accuracy Scale (DANVA)21 in the same ALSPAC dataset. Theirs did not find any associated variants, and nor did they identify a heritable component from SNP based heritability. However, a potential confound in Coleman et al.’s analyses is population stratification, which was not fully taken into consideration. Moreover, other layers of analysis, such as gene based association and transcriptome-wide association, provide additional opportunities for identifying signals that may be otherwise not seen in GWAS.In contrast, Coleman and colleagues21. Specifically, we examined the common genetic architecture of EE among 8-year old males and females evaluated using an established EE paradigm and genotyped and imputed to Phase 1 version 3 of the 1000 Genomes Project22. Further downstream analyses, including gene based and transcriptome-wide association along with polygenic risk score predictive analysis, were also undertaken.Therefore, in this current study ALSPAC data were used to investigate emotional empathy (EE) using the Diagnostic Analysis of Nonverbal Accuracy Scale (DANVA)The total sample comprised 4,780 children  with a mean age at time of assessment of 103.6 months (SD: 3.7 months). There was no difference between the mean ages of males and females  104 months (3.8); females: mean (SD) 104 months (3.6). The mean (SD) IQ  assessed at the same age was 105.1 (16.4) for the complete sample, with no sex difference  105 (16.9); females: mean (SD) 105 (15.7).3 distribution). A cubed transformation of the raw scores improved distributional properties towards normality. DANVA3 was therefore used as the trait measure in all subsequent analyses. Both age and IQ were significantly correlated with DANVA3 scores, and DANVA3 scores differed significantly between males and females . Our principal GWAS analyses therefore examined DANVA3 residualized for the effects of age, sex and IQ. As a corollary, and in order to be able to directly compared results to those of Warrier et al.18, we also conducted analyses residualizing for only age and sex.The total raw DANVA score (max. 24) did not follow a normal distribution  was generated (lambda-GC = 0.99). This intercept of less than 1 is consistent with the absence of population stratification. A SNP heritability of 3.25% (SE 8.8%) was obtained. The results of the GWA analysis for emotional empathy across all autosomes, as measured by total DANVA3 score residualized for the effect of sex, age and IQ, are depicted in Fig. −8 level. In the female-only analysis the most significant locus identified was on chromosome 1 . This SNP lies in an intergenic region. The next most significant loci were in the male only analysis on chromosome 17 , chromosome 4  and chromosome 7 . None of these loci overlap with the Warrier et al. cognitive empathy loci16. Additionally, none overlapped the top SNPs from the Coleman et al. study. All SNPs with p < 1 × 10−6 are provided in Supplementary Table All individuals were of European ancestry according to our population stratification analysis (see methods). We further investigated for confounding using LDSC3 score residualized for the effect of sex and age. The results are shown in Supplementary Fig. et al.16 Additionally, none overlapped the top SNPs from the Coleman et al. study. All pruned SNPs with p < 1 × 10−6 are provided in Supplementary Table We also undertook a GWA analysis of total DANVAet al.’s top 10,000 SNPs to examine concordance with our own analyses. We first LD pruned these SNPs using LDlink’s SNPclip option based on the 1000 genomes phase 3 (version 5) dataset24. We then examined the distribution of the p values for these same SNPs from our ALSPAC GWAS, and tested those p values for deviation from a uniform distribution using the Kolmogorov-Smirnov test. Our data were consistent with uniformity, indicating lack of directional concordance .We additionally investigated direction of effects for Warrier 25. No gene passed the genome-wide threshold for significance (Bonferroni 0.05/15056 = 3.3 × 10−6) , 11  and 17 . This gene was also identified by the intronic variant signal at rs2032753 in the GWAS. The other gene was from the corollary female only analysis  on chromosome 12 .We next undertook a gene-based association test for a total of 15,056 autosomal genes using MAGMA [v.1]et al.26. Two signals from the non-stratified analysis residualized for age, sex and IQ were significant at the Bonferroni corrected level of significance (0.05/2057 = 2.4 × 10−4), with under expression of the genes positively associated with emotional empathy . . AL118508, also on chromosome 20, is a non-protein coding pseudogene, whose coordinates lie within those of AL118508, which explains its signal. Neither are obvious candidates for involvement in the brain processes that underlie emotion recognition and its developmental pathways. Given the recent suggestive findings of new potential mechanisms involved in ASD, however, these genomic regions should be further scrutinized35.We undertook a genome-wide association study of emotional empathy as measured by emotion recognition skills in 4,780 8-year old children. We failed to find any genome-wide significant signal in either our unstratified analysis or analysis stratified according to sex. A gene-based association analysis similarly failed to find any significant loci. In contrast, our TWAS identified two significant loci in the unstratified analysis, residualised for the effects of age, sex and IQ. MYO1D, identified in both GWAS and gene-based association for the male only analysis. This gene encodes a widely expressed protein which is also strongly expressed in the developing brain36 and is in a region that has previously shown evidence of linkage to the neurodevelopmental disorder autism spectrum disorder37. This is particularly significant as ASD is known to be associated with impairments in the ability to decode emotional expressions in faces10.Among the non-significant findings, signals are either intergenic or overlapping genes that are not strongly brain expressed or implicated in brain development. The one exception is et al. in their GWAS study of cognitive empathy on the same sample, nor with the Coleman et al. study in the same sample. This may be unsurprising since these studies are likely underpowered based upon their sample sizes. Indeed, the importance of sample size, and the approximately linear relationship between number of identified associated loci and the size of the sample, has been previously demonstrated38. GWAS studies of complex disease traits have consistently demonstrated the small effect size at individual associated variants. For behavioural traits more generally, the effect sizes are smaller, and the impact of other factors such as everyday experiences  may be large. As such, larger sample sizes will be needed to fully realise the allelic spectra of these traits38.None of the signals identified in our study overlap those of Warrier The largely negative findings from our gene-based and TWAS analyses may similarly be explained by sample size, but other factors intrinsic to the methods themselves may also be relevant. For example, although we restricted our gene-based association analysis to SNPs within each gene, the method does allow extension of the gene’s coordinates upstream and downstream to incorporate surrounding SNPs if there is a good rationale for doing so. Similarly, the analysis can be restricted to only certain SNPs within each gene if, for example, this is based on known functionality of certain SNPs. The method itself uses pruned principal components such that some variance is lost, albeit only a very small amount (0.1%).39.A lack of overlap between the genetics of different aspects of social cognition such as emotional and cognitive empathy may also be expected in light of the functional neuroimaging evidence of regional modularity. In particular, these imaging studies have identified different structures mediating simple and complex emotion recognition. Although there is great interest in using non-human primates for studying brain disorders, particularly in view of their highly developed social behaviour, there are currently no models that have examined genetic association or gene disruption in social behaviouret al.’s studies of cognitive empathy used images of eyes which form part of a standardised measure of cognitive empathy. Extracting the complex emotion depicted in only the eyes might be different from the same information from the whole face. Similarly, static images may involve different cognitive processes than animated images, and social information portrayed in abstract images or cartoons may involve yet other processes. From a methodological point of view, unravelling these elements into more fundamental, orthogonal dimensions of social cognition is a challenge. Similarly, to ensure ecological validity tasks must also necessarily involve real life scenarios which tends to undermine task simplification. This dilemma in the biological research of social cognition will require more sophisticated methods of acquiring data, such as eye tracking which does overcome some of the aforementioned limitations. Similarly, brain imaging phenotypes may offer the opportunity for proxy measures of cognitive phenotypes at the psychological level40.Moreover, in addition to those brain processes involved in different aspects of social cognition, the methodology of different study designs will require the recruitment of additional processes in decoding the information presented. For example, one of Warrier et al.’s, which essentially is based on the same trait in the same sample. There are many relatively arbitrary decisions that need to be made when undertaking GWAS. On consideration of such factors, we note some striking differences between the two studies 21, which includes subtests to measure the ability to decode facial expressions. All participating children were invited to complete this at age 8 years . Please note that the study website contains details of all data that is available through a fully searchable data dictionary and variable search tool . Ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees, and the research carried out in accordance with the guidelines of both committees. Written informed consent was obtained from parents or a responsible legal guardian for the child to participate. Assent was obtained from the child participants where possible.The Avon Longitudinal Study of Parents and Children (ALSPAC) is a longitudinal birth cohort with a sampling frame of all pregnant women living in the Avon region of the UK with expected delivery date between 1The DANVA is a series of computer administered assessments that measure the ability to decode nonverbal information, including facial expression and tone of voice. The ‘faces’ subtest comprises 24 photos of child faces, each showing one of four emotions: happy, sad, anger or fear. Each photo is presented on a computer screen for 2 seconds, during which time the child is prompted to respond whether they think the child portrayed is happy, sad, angry or fearful by clicking on the appropriate word presented on the screen below the image. Comprehension of meaning of the four words, ability to read them and understanding of the task itself is checked by an examiner before the child starts the assessment. From among those who completed the task , 1,139 involved the tester recording the response on a datasheet . For the purpose of the current study, all unrelated children who completed the DANVA were included and then filtered to leave only those individuals with a maximum of one DANVA item missing. A sample of 4,919 children remained at this stage. As discussed subsequently, after further filtering based on the results of our population stratification analysis, a final sample of N = 4,780 children remained. All children also underwent IQ testing using the Wechsler Abbreviated Intelligence Scale .GWAS data were generated by Sample Logistics and Genotyping Facilities at Wellcome Sanger Institute and LabCorp (Laboratory Corporation of America) using support from 23andMe. Genotyping was performed using the Illumina HumanHap550 quad chip genotyping platform. The resulting raw genome-wide data were subjected to standard quality control methods as described in the unpublished ALSPAC quality control document made available to researchers using these data. Individuals were excluded on the basis of gender mismatches; minimal or excessive heterozygosity; disproportionate levels of individual missingness (>3%) and insufficient sample replication (IBD < 0.8). Population stratification was assessed by multidimensional scaling analysis and compared with HapMap II (release 22) European descent (CEU), Han Chinese, Japanese and Yoruba reference populations; all individuals with non-European ancestry were removed. SNPs with a minor allele frequency of <1%, a call rate of <95% or evidence for violations of Hardy-Weinberg equilibrium (P < 5E-7) were removed. Cryptic relatedness was measured as proportion of identity by descent (IBD > 0.1). Related subjects that passed all other quality control thresholds were retained during subsequent phasing and imputation. 9,115 subjects and 500,527 SNPs passed these quality control filters.43 which utilises relatedness during phasing. Phased version of the 1000 genomes reference panel  were obtained from the Impute2 reference data repository . Imputation of the target data was performed using Impute V2.2.244 against the reference panel , using all 2,186 reference haplotypes (including non-Europeans). In total, 27,449,291 SNPs were tested .ALSPAC mothers were genotyped using the Illumina human660W-quad array at Centre National de Génotypage (CNG) and genotypes were called with Illumina GenomeStudio. 477,482 SNP genotypes in common between the sample of mothers and sample of children were combined. SNPs with genotype missingness above 1% due to poor quality  were removed, and a further 321 subjects were removed due to potential ID mismatches. This resulted in a dataset of 17,842 subjects containing 6,305 duos and 465,740 SNPs (112 were removed during liftover and 234 were out of HWE after combination). Haplotypes were estimated using ShapeIT (v2.r644)et al. (2016) QC’d 1000 Genomes phase 3 data as ref. 45. All non-imputed (i.e. Illumina) SNPs that are shared between ALSPAC and 1000 Genomes across the genome were used to investigate population structure after LD pruning . After LD pruning, GCTA [v1.9.1]46 was used to generate a genetic relationship matrix which was in turn employed by GCTA to generate principal components. The first two eigenvectors thereby generated are visualized in the Supplementary Fig. et al. QC’d 1000 Genomes). Although the sample appears to cluster with the 1000 genomes EUR samples, we further investigated this by repeating the population stratification analysis against the EUR only 1000 genomes sample  and confounding bias (intercept minus 1). With true polygenicity, the intercept approaches 1, but with population stratification the intercept shows positive deviation.LD score regression (LDSC)47 [v2.5.2]. The total DANVA score, indicating number of correct responses, was used as the quantitative trait of interest, with age and sex as covariates. DANVA scores were first cubed to normalize residuals 26. Briefly, gene expression levels are imputed into the dataset based on the results of reference data in which gene expression and genotyping have both been performed. This imputation is based on the weights that have been assigned to SNPs in the reference data using a liner predictive model. Association can then be tested between trait and imputed expression. In this way, this method essentially examines how strongly variants associated with expression are also associated with the trait, and under certain circumstances has greater power than traditional SNP-based GWAS. We used GTEx whole blood (release V.6)17 for reference data using pre-computed weights by the FUSION development team.A transcriptome-wide association (TWA) analysis was implemented in FUSION [V.1]48. Results from published GWAS studies of schizophrenia27, bipolar disorder29, educational attainment30, cognition30, ASD27 and obsessive-compulsive disorder31 were used for calculating scores. All scores were generated using the default average score statistic, and subsequent trait regression was performed with sex as covariant as with all other analyses. Clumping was performed in PRSice using the default r2 of 0.1 and 250 kb windows. Default PRSice p-value thresholds were used, and permutation using the default 10,000 iterations was used to generate an empirical p-value.Polygenic Risk Scores were calculated using methods implemented in PRSice-2Supplementary Material.Supplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3


This study aimed to improve nitrogen utilization and alleviate the inhibition of straw decomposition during early tillering and the growth of paddy after straw return. Specifically, three different nitrogen fertilizer (base fertilizer) application methods were tested under full straw return: applying the compound fertilizer once (J1), applying the compound fertilizer twice (J3) and applying the ammonium carbonate fertilizer plus compound fertilizer (J2). Full straw return without fertilizer (CK1) and no straw return without fertilizer (CK2) were used as the controls. The results showed that treatment with ammonium carbonate fertilizer combined with compound fertilizer (J2) significantly enhanced straw decomposition, light interception and dry matter accumulation at an early stage of tillering, but reduced tiller occurrence at a late tillering stage. Grain yield was affected due to reduced dry matter accumulation, nitrogen use efficiency and number of effective panicles. There were no significant differences in rice growth, nitrogen use efficiency and grain yield between the one-time or two-time compound fertilizer application methods. In contrast, treatment with ammonium carbonate fertilizer combined with compound fertilizer (J2) under full straw return effectively improved straw decomposition and accelerated the return of green and tillering. In addition, the proportion of ammonium carbonate fertilizer affected the nutrient utilization efficiency and yield at later stages. As natural supplements containing valuable nutrients and organic carbon (C), crop straw is often incorporated into soils in sustainable agriculture . The incHowever, straw return can bring about a series of agricultural problems. For example, straw can immobilize nutrients from soil and fertilizers, which inhibit the early growth of rice ,3. This The rice variety was Quanliangyou 681, which was provided by Hubei Quanyin High-tech Seed Industry Company Limited. Ammonium carbonate, compound fertilizers and urea were obtained from Hubei Xinshengyuan Biological Engineering Company Limited, Xinyangfeng Agricultural Technology Company Limited and Hubei Qianjiang Jinhua Run Fertilizer Company Limited, respectively.−1, an alkaline nitrogen content of 90.7 mg kg−1, an available phosphorus content of 23.9 mg kg−1 and a fast-acting potassium content of 158 mg kg−1. The experiment used a randomized block design. The detailed experimental design is shown in 2O5:K2O = 15%:15%:15%) and urea (46% N); N was applied at 195 kg hm−2 rate in this study, N fertilizer was applied at the basal, tillering ; panicle initiation stage in a ratio of 5:3:2. Base fertilizer was applied 5 days before transplantation and during transplanting. The content of P2O5 and K2O in each treatment was adjusted to 97.5 kg hm−2 by using calcium superphosphate (16% P2O5) and potassium chloride (60% K2O); both calcium phosphate and potassium chloride were applied during transplanting. Each experimental plot was 56 m2 in area with a 30-cm-wide and 20-cm-high ridge covered with plastic film to prevent water and nutrient penetration between contiguous plots. A total of 15 plots were used in this study.From 2017 to 2018, the field experiment took place in the Agricultural Science and Technology Industrial Park of Yangtze University, Huazhong Agricultural High-Tech Industrial Development zone, Jingzhou City, Hubei Province, China . In 2017, only the grain yield was measured, and in 2018, the grain yield and other indices were measured. The soil type was light loam (Kakingski soil texture classification system); the basic physical and chemical properties of the soil in the depth range of 0–30 cm were as follows: a pH of 8.05, an organic matter content of 18.75 g kgRice stem and tiller numbers: The number of seedlings was recorded after transplantation; ten representative plants were selected to calculate the average number of stems and tillers in each plot. The number of rice tillers was investigated every 4 days until the number of tillers stabilized.The SPAD value of rice leaves: The chlorophyll analyzer SPAD-502 Plus (Konica Minolta Japan) was used to determine the tillering stage, booting stage, full heading stage and maturing stage; the first complete leaf from the main stem was measured. The average of the upper 1/3, middle 1/2 and lower 1/3 of the leaf was taken as the SPAD value for the plant. Each treatment was repeated five times.T) was measured at the top of the canopy (30 cm above the top of the plant). The PAR (PARR) was measured at the top of the canopy vertically. The incident PAR (PART) was measured at the base of the plant canopy.Canopy photosynthetically active radiation (PAR) transmission parameters: The tillering, booting, full heading and maturing stages of rice were determined at 11:00–13:00 on a sunny day using the AccuPAR plant canopy line analyzer (Decagon USA) The incident PAR  and buried (5–10 cm) into each plot in the field before transplantation. The mesh bag was removed from the plots, washed with water, dried, and weighed at 5 d, 20 d, 35 d, 50 d, 65 d, 90 d and 120 d after transplantation, and each treatment was repeated three times .The dry matter accumulation and total nitrogen content: At tillering, booting, full head and maturing stages, five plants were selected according to the average number of stems and tillers per treatment, killed at a temperature of 105 °C and dried to constant weight at 80 °C. The dry matter weight was measured and repeated three times. The mature samples were crushed and the nitrogen content in each plant was determined by the ECS 4024 CHNSO Classic Analyzer . This procedure was repeated three times.2 was selected from the central part of each plot for yield measurement. Ten rice plants were selected to investigate the average effective panicle number. At the same time, five plants were selected to determine the number of grains per ear, seed setting rate and 1000-grain weight.Yield and yield components: After the maturation of rice, an area of about 5 mp ≤ 0.05).The data were analyzed and plotted using SPSS 17.0 and ORIGIN 2019 software packages. The treatment means were compared using the least significant difference (LSD) test at a probability level of 0.05  / dry weight of initial straw × 100%T / PARITr = PARR / PARIRe = PARI-PART-PARR) / PARIIn = , reflectance rate (Re) and interception rate (In) were calculated as follows:I, PART and PARR are the incident PAR (µmol m−2 s−1) at the top of the canopy, the incident PAR (µmol m−2 s−1) at the bottom of the canopy and the reflected PAR (µmol m−2 s−1) at the top of the canopy.In the formulae, PARThe apparent nitrogen use efficiency (ANUE) was calculated as follows: /nitrogen application rate × 100%.−1) was calculated as follows: (grain yield in application plots with nitrogen − grain yield in application plots without nitrogen)/nitrogen application rate.The agronomic use efficiency of nitrogen fertilizer  = (grain yield in application plots with nitrogen − grain yield in application plots without nitrogen)/.The physiological use efficiency of nitrogen fertilizer  was calculated as follows: grain yield/nitrogen application.The nitrogen partial factor productivity . However, the light interception rates of the panicle and mature stages after J3 treatment were lower than after J1 treatment. In the absence of fertilization treatment, the light interception rate of CK1 was lower than that of CK2 in each period and the difference was significant at the booting stage.Changes in PAR transmission parameters of rice canopy under different fertilization methods with time are shown in Changes of tiller number of rice with time under different fertilization methods are shown in Changes in dry matter accumulation of rice with time under different fertilization methods are shown in Nitrogen use efficiency of rice under different fertilization methods is shown in Rice yield and its composition under different fertilization methods are shown in As shown in Straw decomposition is beneficial for nutrient absorption by crops and facilitates crop growth in the long term . PreviouDuring the early stages of rice growth, nitrogen deficiency occurs in plants due to the “contending for nitrogen” phenomenon between microorganisms and plants, which can cause growth inhibition ,23. In tThe efficiency of nitrogen use is an important indicator of nitrogen absorption by rice plants, and it serves as a reference for the application of nitrogen fertilizer in rice fields . In thisThe SPAD value and photosynthetically active radiation (PAR) are key determinants for plant growth and development. The SPAD value represents chlorophyll content in rice leaves and indirectly reflects the photosynthetic capacity of plants ,26,27. NAmmonium carbonate, as a fast-acting nitrogen fertilizer, can provide sufficient nitrogen nutrients for microbial maturation at the seedling stage, promote the rapid maturation of straw, accelerate the release of straw nutrients, provide quick-acting nutrients for plant growth and reduce the inhibition of the early growth of rice plants. However, the effects of ammonium carbonate fertilizer are very fast and easily lost, resulting in insufficient nutrients at later stages. In this study, the tillering speed, dry matter accumulation and light interception rate in the early stage of J2 treatment had certain advantages over other treatments. And the straw decomposing rate of J2 treatment was higher than other treatments, which also showed that the early stage of J2 treatment had enough nutrients to support crop growth and improve the crop’s light interception rate. However, these indices and SPAD value and nitrogen utilization rate of J2 treatment showed a certain disadvantage compared with other treatments. These results provided good evidence support for the above speculation. Future studies should focus on optimizing the timing of application and ratio of ammonium carbonate/compound fertilizer to further promote straw decomposition and rapid seedling emergence, as well as to ensure yield gain.After full wheat straw was returned to the field, the combination of 25% ammonium carbonate and 25% compound fertilizer as the base fertilizer was conducive to straw decomposition and the rapid seedling emergence of rice.


Gallid herpesvirus 1. ILT prophylaxis measures often include using live attenuated vaccines. The live attenuated vaccine can, however, lead to the formation of new strains of ILTV as a result of vaccine reversion and recombination with field strains. Therefore, this study was performed to explore the multilocus variation of ILTV strains of field and vaccine origin. Samples were tested from two distinctive geographical areas in Iraq as little is known about the ILTV genetic diversity within these areas.Infectious laryngotracheitis virus (ILTV) causes a highly pathogenic respiratory disease that affects poultry. It is also known as The polymerase chain reaction method was utilized to generate sequencing templates of six highly polymorphic genes, including UL54, UL52, gB, ICP18.5, ICP4, and gJ in the layer chicken sample (n=15). The Western blotting technique was also employed to detect and estimate the native molecular weight of gE.The results revealed an important degree of genetic relatedness between the field and vaccine strains across all genes. In addition, gE was found to be expressed natively at 49 kDa.The findings of this study may be used to improve the production process of the vaccine for more effective ILT prophylaxis and could further the understanding of epidemiologists and immunologists to better control ILT in the future. Gallid herpesvirus 1 (GaHV-1). It is an alpha herpesvirus that belongs to Iltovirus genus [Infectious laryngotracheitis (ILT) is an avian respiratory disease caused by us genus . The genus genus . There aus genus . ILTV haus genus . These tus genus . Viral gus genus .Live attenuated vaccines can be produced from chicken embryo origin (CEO) or tissue culture origin . HoweverRestriction fragment length polymorphism (RFLP) has been used to discriminate ILTV strains. However, it is relatively expensive and labor-intensive in comparison with real-time polymerase chain reaction (PCR), but the latter and former methods could be combined to provide a better result . RecentlTherefore, this study was performed to explore the multilocus variation of ILTV strains of field and vaccine origin. Samples were tested from two distinctive geographical areas in Iraq as little is known about the ILTV genetic diversity within these areas.The Ethics Review Board of Al-Qadisiyah College of Agriculture and College of Veterinary Medicine approved this study design.× g for 1 min while the pellet (by QIAamp DNA tissue and blood kit) was used for DNA extraction according to the manufacturer’s recommendation.The necropsied tracheal tissue from layer chickens (n=15) was collected from private laboratories in two different regions in Iraq (the North territory and the South region) . This is2+, as well as 1 µM of each, forward and reverse primers, in addition to 400 ng of template DNA (® Safe DNA Gel Stain (1×)  [Analysis of ILTV sequences was conducted using PCR in a total reaction volume of 25 µL consisting of 12.5 µL of 2× Qiagen Multiplex Master Mix and includes Hot Start Taq DNA polymerase with a concentration of 3 mM Mglate DNA . The amp® Fluorometer recommendations, then sequenced at Source Bioscience  by Sanger Sequencing service. The generated sequences were aligned at BLASTn (http://www.ncbi.nlm.nih.gov/) to confirm the targeted sequencing. Thereafter, they were aligned by BioEdit software  for pairwise alignment checking. Subsequently, the phylogenetic tree of each gene was generated using MEGA7 software, version 7.0.21(https://www.megasoftware.net/).All PCR amplicons were purified according to the QIAquick PCR Purification kit manual recommendations  and measured according to the Invitrogen Qubit™ BCA Protein Assay Kit, UK) and heated for 5 min at 95°C. Thereafter, 20 µg of denatured protein samples were electrophoresed in SDS polyacrylamide gel electrophoresis (12%) with 5 µL of protein ladder  using 1× electrophoresis running buffer, containing 192 mM glycine, and 50 mM Tris 0.1% (w/v) SDS pH 8.3, in a vertical gel electrophoresis tank  at 200 V for 2 h at room temperature (23°C) [2) and visualized by ChemiDoc™ Touch Imaging Systems  [The Western immunoblotting served to explain the native molecular size of gE. First, whole protein lysates were extracted by cutting the tissues into small pieces (150 mg) then adding 500 µL of RIPA buffer  followed by adding 300 µL of a protease inhibitor cocktail  . The lysates were sonicated with ultrasonic apparatus VCX130, Sonic) for 7 s. The supernatants were collected, quantified using bicinchoninic acid assay  .All the targeted genes including UL54, UL52, gB, ICP18.5, ICP4, and, gJ were successfully amplified at molecular sizes 544, 473, 638, 627, 603, and 367 bp, respectively, as shown in The results showed a considerable association within the ILTV strains in the two geographical regions of Iraq (North and South) as some samples exhibited a close phylogeny. More importantly, the results show a degree of relatedness of the field and vaccine strains across all genes . Some strains, originating from both geographical regions, revealed genetic interference with a locally used vaccine strain known as Laryngo-Vac .Analysis of the ICP18.5 gene demonstrated a clear genetic interference between the sequences of the North and South regions. These sequences were not closely associated with the vaccine and reference strains . A similIn the ICP4 gene, all samples were genetically split into two groups; most of them were clustered within the vaccine and reference strains due to high genetic similarity . A similAnalysis of gB gene sequences demonstrated a distinctive association between the field samples that originated from both North and South geographical regions. However, two samples from the South (sample numbers 13 and 15) revealed a similarity with two Northern strains (sample numbers 1 and 8). Similarly, sample numbers 14 and 11, from the Southern region, were branched from many of the Northern samples. Notably, the results exhibited a genetic interference between the vaccine strain and sample numbers 10 and 12, both of which originated from Northern and Southern regions .In the gJ gene, a clustering pattern was noticed within the samples and where they originated from. The samples from the Northern region branched off and stayed separated from the Southern samples, with the exception of two samples from the South (sample numbers 11 and 14) . As obseThe native gE glycoprotein expression was observed to be approximately 49 kDa. However, the heat-denatured positive samples exhibited a slightly different result, with approximately 48 kDa in size due to the physical denaturation of the glycoprotein .This study demonstrated that there is significant genetic variability between strains of ILTV. The results revealed a high level of relatedness between field outbreak strains and vaccine strains with different degrees of relatedness depending on the gene being assessed.A vaccination program immunizing against ILTV has not officially been adopted by the federal government of Iraq  because It has been found that field strains of ILTV from Iraq are strongly related to vaccine strains, indicating that continued use of current attenuated vaccines leads to recombination and genetic diversity in Iraqi’s farms. Numerous studies have shown that most of the global outbreaks are the result of CEO vaccine recombination. For instance, the genetic analysis conducted by RFLP revealed potential similarities between filed strains of a Korean outbreak and CEO vaccine, indicating that vaccine recombination was responsible for the epidemic . In AustThe current issue results from developing and using a vaccine from a foreign strain. This is most likely what leads to the introduction of new ILTV genes into a region. For instance, a US CEO has been found to be the original wild ILTV recombination from a strain Italy . All curThe second phase of this study involved detecting the ILTV-gE from field strains to confirm the ILTV infection. This allowed us to distinguish between vaccinated and infected samples thus, testing only the infected samples by PCR for genetic analysis. Moreover, explaining at which molecular weight the native gE is expressed could be utilized to evaluate the possibility of using this glycoprotein as a vaccine candidate. Our results exhibited gE expression at 49 kDa. Eventually, this glycoprotein had been expressed  as a tagAs in other alpha herpesviruses, gE and gI are expressed as a heterodimer and have been found to have a pivotal role in cell-cell infection, while deleting this complex leads to loss of ILTV virulence .Our findings from this study may have an impact on vaccine production for ILTV prevention in the future. Utilizing the multilocus genetic profile of ILTV in the field strain could help epidemiologists and immunologists develop a more effective vaccine strategy. The current control approach for ILTV needs to be reassessed to avoid further outbreaks and spreading.MHAA designed and carried out the study. MHAA drafted, revised and approved the final manuscript.