The attrition rate was high, mainly caused by infections in this instrumentalized animal model. The effect of renal denervation was confirmed by pathology (nerve damage), biochemical parameters and blood pressure measurements. The conclusion is that this model could be used in the assessment of different therapies aimed to modulate the influence of the autonomic nervous system on hypertension if the high mortality rate is reduced with a less invasive monitorization system.High blood pressure  is a major public health problem as far as it has a high prevalence in the adult population and it entails significant cardiovascular and cerebrovascular risks. New ways of treating hypertension include catheter-based renal denervation, i.e., modulation of the autonomic nervous system that has a crucial role in the regulation of blood pressure. We have designed a hypertensive animal model to test the efficacy and safety of this kind of treatment. After the induction of mineralocorticoid mediated hypertension in minipigs, we have analyzed the durability of hypertensive state, the response to drugs and the effects of renal denervation obtained with a radiofrequency catheter (Symplicity® system. Norepinephrine (NE) renal gradients were determined before and 1 month after RDN. Renal arteries were processed for histological  and immunohistochemical  analyses. BP significantly rose after DOCA implants. Six animals died prematurely, mainly from infectious causes. The surviving animals showed stable BP levels after 5 months. One month after RDN, nerve damage was showed in three animals, with impedance drop >10%, NE gradient drop and reduction in BP. The fourth animal showed no nerve damage, impedance drop <10%, NE gradient increase and no change in BP. In conclusion, the minipig model of DOCA-induced HTN is feasible, showing durable effects. High mortality should be addressed in next iterations of this model. RDN may partially offset the DOCA-induced HTN. Impedance drop and NE renal gradient could be markers of RDN success.New-generation catheters-based renal denervation (RDN) is under investigation for the treatment of uncontrolled hypertension (HTN). We assessed the feasibility of a large animal model of HTN to accommodate the human RDN devices. Ten minipigs were instrumented to measure blood pressure (BP) in an awake-state. HTN was induced with subcutaneous 11-deoxycorticosterone  implants. Five months after, the surviving animals underwent RDN with the Symplicity Uncontrolled hypertension (HTN) has been described as a multifactorial phenomenon involving multiple biological mechanisms; the hyperactivity of the sympathetic nervous system plays a paramount role in its onset, maintenance and progression . To dateIn this preclinical setting, large-scale studies have been performed to determine the safety of delivering RF therapy to the renal nerves. Immediate  and chroIn this study, our goal is to develop a large animal model of hypertension that would be reliable, reproducible, and affordable to industry and academia and that would accurately represent the clinical scenario. We analyze the feasibility of a minipig model of 11-deoxycorticosterone (DOCA)-induced HTN, assessing the persistence of HTN and the effect of RDN on the DOCA-induced HTN. However, the high mortality should be addressed in next iterations of this model.The care and use of animals for this preclinical study was reviewed and approved by the Ethics Committee of the Consejería de Agricultura y Ganadería  prior to the start of the study. The treatment of the animals was in accordance with relevant site procedures, which utilized the local regulations outlined in the RD 53/2013, 1 February, which defined basic standards for the protection of animals during experimentation and other scientific purposes, such as education, and European Directive 2010/63/EC and the conditions specified in The Guide for Care and Use of Laboratory Animals, Eighth Edition  as a guide. The study design is presented in The study was implemented using ten 3-month-old female minipigs weighing 20 ± 3 kg, which were provided by Granja Experimental IMIDRA N Registro EX 013-C. The pigs, housed individually in a special room with a suitable temperature (23 ± 1 °C) and humidity (50 ± 5%), were maintained on a standard chow diet.Following the acclimation period (at least 1 week), all animals underwent the placement of a blood pressure (BP) measurement catheter. On the day of catheter placement, the animals were administered 0.35 mg/kg of midazolam and 5 mg/kg of ketamine intramuscular. After the level of sedation was verified to be adequate, a dose (1–2 mg/kg) of anesthetic propofol was administered by slow intravenous infusion. It was inserted through an endotracheal tube and connected the animals to volume-controlled mechanical ventilation, maintaining anesthesia with sevoflurane at 1.5–2%. Analgesia was procured with intramuscular buprenorphine 0.01 mg/kg. The permanent intra-arterial catheter  was positioned initially in the carotid artery; however, catheters were placed in other locations and sometimes moved to the contralateral location or remotely  to ensure proper arterial access. The catheter was tunneled subcutaneously, proximally to an external location that could be easily accessible to the staff and not accessible to the animals to produce damage to the catheter. Following the animal after recovery, multiple BP reads per day over a period of 3 days were acquired while the animal was awake. This measurement was considered baseline for the animal. For a period of 60 days, the BP was monitored and averaged A,B. AfteAfter 5 months of the initial hypertension induction procedure, RDN ablation therapy was performed according to common protocols of catheter-based renal denervation . In brieThe Symplicity™ Renal Denervation System  is a highly flexible and 6 French guide catheter-compatible device with unique single point of contact energy delivery capabilities. Symplicity™ is designed to deliver low level RF energy through the wall of the renal artery for renal denervation. The system consists of two major components: a sterile single use ablation catheter and a proprietary generator, which delivers RF energy sufficient for ablating sympathetic nerves adjacent to the treatment site ,17,18. TComplete necropsy and histopathological study were conducted on all animals that died during study to determine the cause of the death. At the end of the follow-up period, the animals were heparinized and humanely euthanatized. Gross pathology was performed in order to evaluate macroscopic changes to the treated kidneys, heart, lungs, liver, spleen, bowel, ovaries, adrenal glands prior to en-block resection of the treated renal arteries, attached kidneys and surrounding tissues.For the extraction of the area of interest, the renal arteries were pressure perfused through the cannulated aorta with a Lactated Ringers solution followed by perfusion fixation with 10% neutral buffered formalin (NBF) solution for approximately 10 min. The treatment area and surrounding tissues that included the kidneys, major vessels, ureters, adrenal glands, renal lymph nodes and any associated soft tissues, were dissected. Tissue explants were immersed into 10% NBF for at least 2 days, supported by a solid cork backing to ensure stability and structural integrity of the tissue block. After fixation, the aorta was bisected to expose the renal ostia. The renal artery and approximately 1.5–2 cm radius of the surrounding retroperitoneal connective tissues was isolated by removing excess tissue to create a roughly 3-cm-wide renal “stump” centered on the renal artery. Equidistant sections were taken from the renal ostium to the renal hilum, resulting in 5–6 cross-sections along the length of the treated renal artery that included associated soft tissues . These transverse sections were examined grossly for any evidence of injury.Tissues were processed routinely for histology and embedded in paraffin blocks. Serial 3 µm sections were stained with hematoxylin-eosin (HE) and Movat pentachrome stains. The histopathology evaluation of renovascular damage included: endothelial cell loss, medial damage , inflammation and adventitial fibrosis. The magnitude of the changes was assessed using a semiquantitative ordinal grading system: 0, none; 1, minimal; 2, mild; 3, moderate; 4, severe. The endothelial cell loss was evaluated by the percentage of circumferential endothelial cell loss and a semiquantitative score was established: 0, 100% of the luminal surface covered by endothelium; 1, endothelial loss <25%; 2, endothelial loss 25–50%; 3, endothelial loss 50–75% and 4, endothelial loss >75% of vessel circumference .l-lysine-coated slides . Sections were dewaxed in xylene and rehydrated in graded alcohol solutions, and they were stained using the Avidin–Biotin complex method or the EnVision(+) method for S100 or TH, respectively. Endogenous peroxidase activity was quenched by incubation in hydrogen peroxide (0.5%) solution in water for 30 min at room temperature. Tripsin 0.1% pH 7.8  or heat antigen retrieval (EDTA/TRIS Tween pH 9) was performed for S100 or TH, respectively. Following this procedure, slides were blocked with 5% goat serum  and then incubated with primary antibodies . The following day, slides were washed 3 times in TBS and incubated with a biotinylated secondary antibody . Formalin-fixed, paraffin-embedded untreated renal artery sections were used as positive controls. The primary antibody was replaced with antibody diluent for negative controls. Labeling was visualized with application of 3-3-diaminobenzidine-tetrahydrochloride (DAB) as chromogen substrate . Slides were counterstained with Harris’s hematoxylin, dehydrated in graded alcohol, and mounted with coverslips. The intensity and distribution of immunostaining can be assessed using a semiquantitative scoring system: 0, no reaction; 1, patchy/very weak reaction; 2, weak to moderate reaction; 3, strong reaction. Grade 2 or lower was considered to be an indicator of nervous damage [Sections were immunostained for expression of S100 for Schwann cells  and tyrosine hydroxylase (TH) for efferent nerve fibers . Serial 3 µm sections of paraffin-embedded samples were cut and mounted on poly-s damage .Morphometric analysis was conducted on the images using Image-Pro Plus software to quantify the following parameters: number of nerves per section (S100), number of damaged nerves per section (TH) and media thickness at and away from the RF contact point.Blood samples were collected through the femoral catheter before RDN and 4 weeks after RDN. Arterial and venous blood samples were kept frozen until analysis at the bioanalytical test site . The entire volume of the blood samples was homogenized with 3/16″ stainless steel ball bearings in a bead homogenizer. Samples were centrifuged and filtered. Calibration standards were prepared in aqueous 10 mg/mL sodium metabisulfite and the calibration range was 0.100 to 100 ng/mL. Stable isotope labeled norepinephrine was added to each sample  as an internal standard. Samples were derivatized followed by solid phase extraction. Samples were then injected onto an HPLC-MS/MS system for quantitation. Analysis was performed under HPLC with an electrochemical .p value < 0.05 as a cutoff for statistical significance.Values are expressed as percentages and as mean ± standard deviation, depending on the type of variable. Differences between means and repeated measurement of values were analyzed using the Student t test and analysis of variance. For multiple comparisons, a post hoc analysis was conducted using the Dunnett method for comparison with the control. Semiquantitative variables were analyzed using the chi-square test or the Fisher exact method. All analyses were conducted with the JMP v13 statistical software package  using a Ten female minipigs were included in this study. In all ten, the BP monitoring catheter was placed and an average baseline arterial pressure was possible to obtain. The DOCA implant was successful in all 10 animals. Following this procedure, the catheter needed to be relocated in multiple occasions due to occlusions or signs of infection at the access sites. Unfortunately, due to catheter complications, six animals resulted on early death or required early euthanasia following veterinary evaluation. Four animals survived to the end of the study. The causes of death were varied: for one animal, during the placement of the BP monitoring, the thoracic aorta was damaged, resulting in an aortic aneurysm and eventual rupture since the animal had a hypovolemic shock that was not resolved. Two animals developed septicemia that was not resolved despite antibiotic regimen, while the other developed endocarditis and valvular vegetations with associated thromboembolism. Two animals were euthanized due to impaired clinical status and one diagnosed with hypovolemia following upper gastrointestinal bleeding (gastric ulcer), and, for another, the source of the loss of blood was not found.n = 6) and some even survived to the initial antihypertensive medication regimen (n = 4), the BPs acquired during hemodynamically stable periods were still used in each according stage. The renal arteries from these animals (n = 12) were still harvested per protocol and evaluated as “non-RDN treated controls” for a total of 20 renal arteries evaluated.The early deaths and early euthanasia animals survived 87.5 ± 49.3 days post DOCA implant placement (from 30 to 146 days). As these animals survived model creation . One month after the surgical placement of the DOCA implant, there was a 20% increase in SAP . The values of diastolic arterial pressure (DAP) followed a similar pattern with baseline values of 98 ± 7 that raised to 130 ± 9 mmHg (p = 0.0001). Following the antihypertensive medication regimen (spironolactone 200 mg daily + doxazosine up to 16 mg daily), there was a significant decrease both in SAP  and in DAP  (n = 4). One month after the RDN procedure, there was an evident decrease of approximately 6% . Three animals showed a reduction ≥ 8 mmHg in SAP, whereas one pig (# 3) showed a slight increase in SAP. The response of DAP to RDN tended to be much more irregular. The baseline systolic arterial pressure (SAP) was possible to acquire in all ten animals. The baseline SAP was 148 ± 12 mmHg (= 0.017) A. FollowThe main renal arteries had an average length of 26.9 ± 9.7 mm between the aorta and the main arterial bifurcation. At baseline, the mean luminal diameter (LD) in the main renal artery was 5.3 ± 0.6 mm . Past the bifurcation, the LD of the branches was 3.5 ± 0.4 mm; the inferior renal branch being on average slightly larger (3.7 ± 0.3 mm) than the superior branch (3.3 ± 0.5 mm). The characteristic image previously described as “notches” following RF treatment delivery was evident in all treated arteries. The notches observed following treatment delivery at baseline were completely resolved at follow up. All arteries displayed undisturbed blood flow pre and post treatment delivery with no evidence of thrombus or intimal dissection.There was no evidence of collateral RF damage to any abdominal viscera following the catheter-based procedure. At gross evaluation, there were no abnormal changes noted in any of the renal arteries. There were no abnormal gross or microscopic pathological changes observed in the renal parenchyma of any kidneys that could be related to the delivery of RF therapy.A total of 47 sections were examined from four animals with eight renal arteries. The location where RF treatment was delivered was evident in the exposed artery by the involvement of the media C,F,I. ThThe percentage of damaged nerves per level was high in the animals # 1, 2 and 4 based on TH immunostaining (score ≤ 2), while almost all nerves showed normal appearance with TH staining = 3 in the animal # 3 .A decrease in NE renal vein–artery difference (gradient) from pre to post procedural levels was observed in three animals . Conversely, the animal # 3 showed an increase in this value. Interestingly, this animal didn’t show nerve damage at the pathological analysis. The NE gradient reduction showed a correlation with the impedance drop and the mean temperature achieved .In this study, the hypertensive pig model induced by low dose sustained-release DOCA implantation is raised as a feasible platform to evaluate renal denervation therapies. The main findings of this study were as follows: (1) the minipig model of DOCA-induced HTN seems to be feasible, showing durable effects; (2) the RDN with RF may partially offset the DOCA-induced HTN after 4 weeks due to substantial nerve damage; (3) a relation may be established between the effectiveness of RF RDN  and procedural parameters ; (4) the catheter-based RF RDN seems to be safe and only mild renal injury is observed; (5) the direct arterial BP monitoring through an indwelling catheter was unfeasible.n = 10 pigs), which is in line with previous reports [Disease animal models have been extensively used in the understanding of arterial hypertension . The dev reports ,31. More reports . The resNowadays, measurement of BP by radiotelemetry has been described and validated for many laboratory animal species. This technique eliminates the need for tethering or restraining the animals, which introduce stress and artifacts during data sampling . HoweverIn the present model, the reduction in BP was related to the nerve injury identified by the reduced immunohistochemical expression of TH, which is considered key evidence of a proper RF nerve ablation . In thisOne of the major unsolved issues of the procedure is how to monitor treatment success intraprocedurally. In the present study, animals with reduced BP showed NE gradient reduction and impedance drop, although there was not a clear relationship between the magnitude of NE reduction/impedance drop and the reduction in BP. However, the case of animal # 3 suggests a potential working hypothesis: no nerve damage was observed in the case when the impedance drop was below 10% during the procedure; in addition, NE gradient did not decrease  and BP response was flat. Be it a technical failure in the RF RDN procedure or an individual abnormal response, the absence of all the studied responses is interesting.Finally, the RF procedure displayed a high safety with none-mild inflammatory response, complete vascular endothelialization without vascular thrombosis, hemorrhage or dissections, similar to the one described in clinical and preclinical trials using radiofrequency energy ,39,40. HThe main limitation of this study was that the creation of the model requires a learning curve and the placement of the blood pressure catheter measurement produces significant technical difficulties and has a direct impact on the clinical outcomes of the animals. We believe that a combination of our suggested animal model with a telemetric measuring technique would decrease infection rates. Another limitation is related to the physiological responses of primary hyperaldosteronism (PHA), that is not the most common form of clinical hypertension. However, the decrease in blood pressure following surgical denervation of a DOCA swine model  supportsThe data obtained in this preliminary study suggest that the DOCA large animal model could be used as an experimental platform of reproducible, long term maintenance of arterial hypertension. We believe that further investigation with proper animal models, such as the one presented in this study, is critical for the advancement of this therapy. Ablations performed in the distal region might improve the effectiveness of radiofrequency RDN. This observation seems to confirm the importance of anatomic and procedural parameters when performing RDN and deserves further investigation in clinical studies. However, the high mortality should be addressed in the next iterations of this model.


The purpose of this study is to compare the difference of clinical efficacy between conventional intraperitoneal chemotherapy and HIPEC, so as to explore the clinical application value and advantages of HIPEC.χ2 test, and quantitative data were processed by t test. P < 0.05, statistical data can be considered statistically significant.A retrospective analysis was conducted on 80 patients with malignant ascites admitted to our hospital from June 2017 to June 2019. The general clinical data and qualitative data of the treatment results of 80 patients with malignant ascites were processed by SPSS19.0 using There was no significant change in vital signs and temperature in the observation group during the treatment, and the difference was not statistically significant.The short-term total effective rate of patients in the observation group was 91.11%, and the short-term total effective rate of the patients in the control group was 40%.There was no significant difference in the incidence of adverse reactions between the two groups of patients.Intraperitoneal hyperthermic chemotherapy combined with intravenous chemotherapy can significantly control malignant ascites and has small adverse reactions, which is worthy of clinical promotion and application. Abdominal or systemic malignant tumors can easily induce diffuse lesion of the abdominal visceral wall and further lead to abnormal increase of peritoneal effusion, which is called malignant ascites clinically . ClinicaThe inclusion criteria were as follows: (1) 16 ≤ age ≤ 85 years; (2) CT, MRI, exploratory laparotomy and/or intraperitoneal free cancer cells confirmed diffuse intraperitoneal implantation metastasis of tumor; and (3) ultrasonography confirmed the abdominal volume of > 3000 ml.The exclusion criteria were as follows: (1) no malignant ascites or abdominal volume < 1500 ml, (2) extensive adhesion in the abdominal cavity, (3) inclusional peritoneal effusion, (4) total intestinal obstruction, (5) severe coagulation dysfunction, and (6) too old and obviously poor physical function.P > 0.05) , partial remission (PR), disease stability (SD), and disease progression (PD). CR means ascites disappeared for more than 4 weeks, PR means ascites decreased by more than 50% and lasted more than 4 weeks, SD means ascites decreased by less than 50% or no change, and PD means increased ascites . Total eχ2 test and quantitative data were processed by t test. P < 0.05, statistical data can be considered statistically significant.The general clinical data and qualitative data of the treatment results of 80 patients with malignant ascites were processed by SPSS19.0 using There was no significant change in vital signs and temperature in the observation group during the treatment, and the difference was not statistically significant Table .Table 2P < 0.01)  Table .Table 3P > 0.05)  Table .Table 4Malignant ascites is a common complication of advanced abdominal and pelvic malignancies . AbdominThe main purpose of treating malignant ascites is to improve the quality of life of the patient, relieve the patient’s pain, and thereby extend the patient’s survival. HIPEC is an auxiliary method used in the treatment of malignant tumors in the abdominal cavity in recent years . A largeIn short, intraperitoneal hyperthermic chemotherapy combined with intravenous chemotherapy can significantly control malignant ascites and has small adverse reactions, which is worthy of clinical promotion and application.


Structural variants are large changes to the DNA sequences that differ from individual to individual. We discovered and quality-controlled a set of 24,908 structural variants and used a technique called imputation to infer them into 35,588 Holstein and Jersey cattle. We then investigated whether the structural variants affected key dairy cattle traits such as milk production, fertility and overall conformation. Structural variants explained generally less than 10 percent of the phenotypic variation in these traits. Four of the structural variants were significantly associated with dairy cattle production traits. However, the inclusion of the structural variants in the genomic prediction model did not increase genomic prediction accuracy.Structural variations (SVs) are large DNA segments of deletions, duplications, copy number variations, inversions and translocations in a re-sequenced genome compared to a reference genome. They have been found to be associated with several complex traits in dairy cattle and could potentially help to improve genomic prediction accuracy of dairy traits. Imputation of SVs was performed in individuals genotyped with single-nucleotide polymorphism (SNP) panels without the expense of sequencing them. In this study, we generated 24,908 high-quality SVs in a total of 478 whole-genome sequenced Holstein and Jersey cattle. We imputed 4489 SVs with R2 > 0.5 into 35,568 Holstein and Jersey dairy cattle with 578,999 SNPs with two pipelines, FImpute and Eagle2.3-Minimac3. Genome-wide association studies for production, fertility and overall type with these 4489 SVs revealed four significant SVs, of which two were highly linked to significant SNP. We also estimated the variance components for SNP and SV models for these traits using genomic best linear unbiased prediction (GBLUP). Furthermore, we assessed the effect on genomic prediction accuracy of adding SVs to GBLUP models. The estimated percentage of genetic variance captured by SVs for production traits was up to 4.57% for milk yield in bulls and 3.53% for protein yield in cows. Finally, no consistent increase in genomic prediction accuracy was observed when including SVs in GBLUP. Whole-genome sequences have facilitated the study of large-scale genomic variations. Structural variations (SVs) are large DNA segments of deletions, duplications, copy number variations (CNVs), inversions and translocations in a re-sequenced genome compared to a reference genome ,2. NumerGenotype imputation is a strategy to infer missing genetic markers of individuals that are only genotyped on lower-density panels from a reference set of individuals genotyped on denser panels . Many stAlthough SVs have been found to be associated with complex traits in various livestock breeds, there is limited knowledge of how much genetic variation is explained by SVs in dairy cattle for complex traits, including milk production traits and fertility. Including SVs in genomic prediction could potentially improve the accuracy of genomic estimated breeding values (GEBV) if SVs explain a proportion of the total genetic variance. In this study, we first detected high-quality SVs using two SV detection programs, Breakdancer  and PindBos taurus assembly UMD3.1 with the Burrows–Wheeler Aligner (BWA) method [https://www.illumina.com/Documents/products/datasheets/datasheet_bovineHD.pdf (accessed on 2 June 2018)). Quality control for imputation followed Erbe et al. [The animal samples and the whole-genome sequence data were available from the 1000 Bull Genomes Project (Run 5) ,25. Ther) method . A large) method . Of thes) method , except e et al. . From thThe process to generate high-quality structural variation (SV) sets is described in Chen et al. . BrieflyFor each of the 478 animals, SVs from the Pindel output (in VCF format) were compared with each of SV sets above using bedtools  to produOne of the main factors that can influence imputation accuracy is the marker density of the reference population. To investigate the impact on imputation accuracy by reference SNP density, three different SNP panels were used as reference panel scaffolds for imputation and combined with the various SV sets: panel 1, SNP genotypes from Illumina BovineHD BeadChip; panel 2, SNP positions from BovineHD BeadChip with genotypes extracted from a whole-genome sequence; and panel 3, SNP genotypes from a whole-genome sequence whose minor allele frequency (MAF) was greater than 0.05 using VCFtools. The description of these reference panels used for imputation is summarised in The combination of reference panels and SV genotypes from the different SV sets is described in A fivefold cross-validation was used to assess the accuracy of imputation. The 478 individuals were randomly separated into five different sets. One set was treated as a testing set, and the other four sets were the reference set. The SV genotypes of individuals in the testing set were masked as missing (coded as 5), whereas all SNP and SV genotypes in the training set were used as the reference group for imputation. This process was repeated five times, testing each set in turn.Based on the performance of the two programs, we generated a list of SVs imputed with an accuracy above 0.5 from the four different sets with panel 2 using Eagle + Minimac3. After merging all four SV sets by 1 bp overlap, a total of 4361 deletions, 60 inversions and 68 duplications were selected. The merged 578,999 BovineHD SNP and 4489 SV genotypes of the 478 whole-genome sequenced bulls were then used as the reference set to impute SVs on 35,568 dairy cattle. Eagle (version 2.3)  was firsy is a vector of daughter trait deviations (DTD) for a trait; n1 is a vector of ones, n is the number of samples and μ is the population mean term; X contained the genotype indicator variable of candidate markers coded as 0, 1, 2 tested one at a time and the breed indicator variable coded as 0 (pure Jersey), 0.25 (¼ Jersey and ¾ Holstein), 0.5 , 0.75 (¾ Jersey and ¼ Holstein) and 1 (pure Holstein); b is the fixed effect vector for the candidate marker and breed; e are residual effects distributed as e ~ N can be used to approximate the number of true positives at the a et al. :(2)FDR= ggplot2.The model was fitted to the data with GCTA v1.26.0 . ManhattThe genomic best linear unbiased prediction (GBLUP) model was used to predict GEBVs ,35 in ASA 10-fold cross-validation was used to evaluate the genomic prediction accuracy in the bull datasets only. The cross-validation procedure was applied to three sets of bulls: 4057 Holstein bulls, 1058 Jersey bulls and 5115 combined Holstein and Jersey bulls. For each dataset, samples were randomly partitioned into 10 mutually exclusive subsets of approximately equal size for each trait. Each of the 10 subsets was in turn used as validation, while the other 9 subsets were combined and used as training set. Genotypes and phenotypic records were available for the samples in the training set, whereas phenotypes were set to be missing for the samples in the validation set. The cross-validation process was repeated 10 times by re-sampling the sets randomly. The accuracy was calculated as Pearson’s correlation coefficient between the predicted GEBV and the DTDs. The validation was conducted on Holstein, Jersey and mixed Holstein and Jersey breed separately.We generated two population SV sets, POP_HOL and POP_JER, and three validated SV sets, TWICE_SEQ, FAM_HOL and FAM_JER . DeletioOverall Eagle + Minimac3 resulted in a larger proportion of SVs with an accuracy above 0.5 than FImpute . Both piThe distribution of imputation accuracy and imputation accuracy versus MAF for deletions using Eagle + Minimac3 are plotted in p < 10−7) for all the production traits with actual p-values 2.2 × 10−13, 2.5 × 10−25 and 1.6 × 10−12 for fat yield (FY), milk yield (MY) and protein yield (PY), respectively. This corresponds to an FDR of 0.04%. With a less stringent threshold (p < 10−5), one additional deletion on BTA5 was significant for MY (p < 9.2 × 10−6) with an FDR of 4.49%. No significant SVs were found for fertility and overall type. Hundreds of SNPs were significant for production traits and a smaller set of 34 SNPs for fertility (p < 10−7). Manhattan plots and Q–Q plots for MY, fertility and overall type are shown in We performed single-trait GWAS, which detected a small number of significant SVs . One delp-value < 10−7) were identified with a corresponding FDR of 0.01%, which was lower than for any individual trait. These significant SVs and their overlap with single-trait analyses are summarised in A multi-trait meta-analysis for all production traits was performed , and fouWe investigated whether the four significant deletions were in high linkage disequilibrium (LD) with significant SNP in the same region to determine whether the SV was likely tagging genetic effects not tagged by SNP. The LD between each deletion and the surrounding SNPs (±1 Mb for the one on BTA14 as it is on the start of the chromosome and ±500 kb for the remaining three) were calculated . The twoThe genetic and phenotypic variance components for each trait were estimated for Holstein and Jersey bulls separately using models with or without SV effects. Overall, the total genetic variance increased slightly by adding SV information for all traits in both breeds. For all traits except fertility, a larger proportion of genetic variance was explained by SVs in Jersey than in Holsteins. The SVs explained up to 3.8% and 7.3% of the total phenotypic variance for fat and milk yield traits in Holstein and Jersey bulls, respectively . While 3We also estimated the variance components combining Holstein and Jersey animals, but for bulls and cows separately. For the three production traits, the SVs explained up to 3.6% and 1.3% of the total phenotypic variance for both bulls and cows, respectively. For fertility, SVs explained 3.8% of the total phenotypic variance in bulls, but almost zero for cows, whereas for overall type the SV variance components in both bulls and cows were very close to zero . In bullThree sets of bulls including 4057 Holstein, 1058 Jersey and 5115 combined Holstein and Jersey were tested using models with and without SV effects to compare genomic prediction accuracy. Mean correlations between GEBV and DTDs for the two models in 10 replicates of 10-fold cross-validation are presented in Imputation of SVs from SNP genotype could be a cost-effective way to genotype SVs in a large number of individuals, at least for SVs that are at reasonable MAF in the reference population. We identified 24,908 SVs including deletions, inversions and duplications from four SV sets, which were then imputed using two pipelines. Overall, Eagle2.3-Minimac3 resulted in higher imputation accuracy than FImpute. SVs from FAM_HOL and TWICE_SEQ had a higher proportion of SVs with high imputation accuracy than the other sets. This result suggested that the sire–son validation is useful to remove false positive SVs as concluded in Chen et al. . A high The comparison between FImpute and Eagle+Minimac3 implied that the latter pipeline can impute SVs more accurately, especially when the SNP markers are very dense . DespiteWe performed GWAS in Holstein and Jersey dairy cattle breeds for both bulls and cows to investigate the associations between phenotypes, including production traits, fertility and overall type, and SNP genotypes as well as SV information. In single-trait GWAS, many SNPs that were found significant for FY, MY, PY and fertility were also previously reported in the literature, whereas none was found for overall type. In terms of SV, two deletions were significant for milk yield, whereas in the multi-trait meta-analysis four deletions were significantly associated with at least one production trait. The significant deletions on BTA5 and BTA14 may be mainly due to the high linkage disequilibrium (LD) with their surrounding SNPs. The LD for the two SVs on BTA6 and BTA20 and the surrounding SNPs was not high, even though there were many significant SNPs on these chromosomes. This may indicate that these SV tag genetic information additional to that of SNP.A list of genes within ±500 kb distance of the four significant SVs  was compA small proportion of the total genetic variance was explained by SVs. The proportion explained seemed to also be variable between traits as well as between bulls and cows. The between trait variation can likely be explained by SVs affecting different pathways, whereas the differences between sexes may be due to sampling from unequal population sizes for bulls and cows. Despite some genetic variance being explained by SVs, genomic prediction did not benefit from adding SVs. While the GBLUP model assumes that the SNP effects follow a normal distribution, other models assume that the SNP effects follow a mixture distribution such as BayesR  or exponIn our study, the most likely reason that no significantly higher accuracy was found in the SNP+SV model is that the set of selected SVs were imputed from SNP arrays. Genomic prediction based on imputed whole-genome sequencing data may have very small or no benefit compared to SNP array data ,49. SNPsOur analyses were based on the UMD3.1 reference assembly and Run5 of the 1000 Bull Genomes Project. More recent runs of the 1000 Bull Genomes Project include a much larger set of Holstein cattle and approximately twice the Jersey samples. This would likely affect imputation accuracy as it would increase the reference dataset. Likewise, the ARS-UCD-1.2 bovine assembly has better resolved repetitive regions of the genome . HoweverThe accurate detection of structural variants from short-read whole-genome sequence and subsequent imputation into animals with only SNP chip genotypes presented a significant challenge. Overall, few SVs were found to be associated significantly with phenotypes, SVs explained a small proportion of the genetic variance, and there was limited influence of SVs on genomic prediction accuracy. Taken together, our findings indicate that the routine application of SVs from short-read sequence data in cattle breeding should not be a priority.


P = 0.001). The rTTE values significantly differed between lesions of neoplastic versus non‐neoplastic origin (P = 0.004). The majority of neoplastic pulmonary masses were supplied by bronchial arteries (63%), while most nonneoplastic pulmonary masses were supplied by pulmonary arteries (78%). The sensitivity and specificity for detecting pulmonary neoplastic masses with rTTE were 63% and 78%, respectively. Enhancement patterns for mediastinal thymomas and lymphomas significantly differed (P = 0.002). Thymomas enhanced heterogeneously in a centripetal pattern (86%), whereas lymphomas typically enhanced uniformly in a centrifugal pattern (75%). Findings indicated that CEUS is a feasible method for characterizing intrathoracic mass lesions in dogs and cats, however, the diagnostic sensitivity for detecting neoplastic pulmonary masses was low.Contrast‐enhanced ultrasonography (CEUS) is increasingly available for veterinary patients, however limited studies describe the use of this method for characterizing intrathoracic mass lesions. The aim of this prospective, observational study was to describe CEUS enhancement patterns for intrathoracic mass lesions in a sample of cats and dogs. Sixty patients  were included. Standardized CEUS examinations were performed for 41 pulmonary masses (68%) and 19 mediastinal masses (32%). Final diagnosis was based on cytology and/or histopathology. Absolute time to enhancement (TTE) values were recorded for the intrathoracic mass lesions and spleen. The spleen was used as a reference parenchymal organ to calculate relative TTE (rTTE) values. Absolute TTE of the spleen and intrathoracic mass lesions differed for dogs and cats ( CEUScontrast enhanced ultrasonographyMImechanical indexrTTErelative time to enhancementSDstandard deviationTTEtime to enhancementTTPtime to peak1Mass lesions are defined as solitary lesions larger than 3 cm in size.Diagnosis can be challenging due to the diverse appearances of nonneoplastic and neoplastic intrathoracic mass lesions in dogs and cats. The use of contrast agents is a common tool in diagnostic imaging. Imaging modalities such as contrast enhanced CT, contrast enhanced MRI, perfusion scintigraphy, and angiography use vascularity patterns for characterization of intrathoracic masses. In recent years, ultrasonographic contrast agents have been used more frequently in veterinary medicine.The purposes of this study were to assess the feasibility of CEUS as a method for characterizing pulmonary and mediastinal mass lesions in dogs and cats, and to determine whether CEUS could distinguish neoplastic from nonneoplastic pulmonary and mediastinal mass lesions. The hypothesis was that nonneoplastic pulmonary mass lesions would differ from neoplastic mass lesions regarding their vascular supply, with neoplastic mass lesions supplied mainly by systemic bronchial arteries. For mediastinal mass lesions, the hypothesis was that, due to the different angioarchitecture, lymphoma and thymoma tumor types would differ in dynamic enhancement characteristics.2All procedures were discussed and approved by the institutional ethics and animal welfare committee in accordance with good scientific practice guidelines and national legislation. Dogs and cats were included in this prospective, observational study when thoracic radiographs showed an intrathoracic mass of at least 3 cm in size and which was available for ultrasound guided sampling during the period of October 2014 to January 2017. Sample size was based on convenience sampling. Exclusion criteria were nondiagnostic samples and therefore no final diagnosis of the mass was performed. The decision for inclusion or exclusion of cases as well as for recording imaging and pathologic findings were made after consensus assessment by two university hospital radiologists (T.R. and K.H.) with one of them experienced in contrast ultrasonography (K.H.). The same authors performed all imaging procedures.®, Alvetra und Werfft GmbH, Vienna, Austria) or under short anesthesia with butorphanol and propofol  if needed. The CEUS examination of the intrathoracic mass was performed first. After a break of 5 min, a second contrast medium injection was performed for the CEUS examination of the spleen. A convex 5–1‐MHz transducer was used with machine settings comparable in both CEUS examinations of the intrathoracic mass and spleen. The CEUS examination was performed with a low mechanical index (MI) setting (0.05–0.11). The focus was set outside the region of interest to not influence the MI. The settings were kept constant for all patients.Ultrasonography of the mass was performed and the origin of the lesion was determined to be either pulmonary or mediastinal. Each patient was examined with an 8–5 MHz curved array transducer . The ultrasound examination was carried out with special emphasis to localization, echogenicity, texture, and contour of the mass, and amount of free fluid in the thoracic cavity. The CEUS procedures and diagnostic biopsies were obtained either under sedation with butorphanol alone  was injected through a peripheral venous catheter placed in the cephalic vein. The catheter was equipped with a three‐way valve and a tube extension. Each contrast injection was followed by a 5 mL saline flush (0.9% NaCl) through the cross connection of the three‐way‐valve.A total of 1 to 1.5 mL  of contrast medium  installed on the ultrasound machine. All measurements of contrast enhancement in the mass were compared to those in the spleen as a parenchymal, systemically perfused reference organ. In two dogs, the liver was used due to previous splenectomy as a result of a ruptured hemangiosarcoma in one dog, and because of splenic nodules of unknown origin in the other.Standardized 5 × 5 mm regions of interest were placed within the lesion where enhancement was most obvious and no large vessels or cysts were located Figure . Signal For pulmonary lesions, the type of vascularization  was determined by comparing the absolute TTE in the lesion to that in the systemically perfused spleen. From this ratio, the relative TTE (rTTE) was calculated (TTE of the lesion/TTE of the spleen = rTTE). The rTTE was defined as “early” if enhancement of the mass was observed before enhancement of the spleen, and “delayed” if it was observed at the same time or later than the spleen. An early rTTE had values < 1 and affected masses were defined as pulmonary arterial vascularized and delayed rTTE had values equal to or higher than 1 and masses were defined as bronchial arterial vascularized.In all patients a fine needle aspiration cytology and/or histology was performed. The type of sampling was determined by the size and location of the lesion and the general condition of the patient.P‐value below 5% (P < 0.05) was considered as statistically significant.Descriptive data were depicted in a contingency table. Patients were divided into seven groups regarding their cytology and/or histology results. Differences between groups in absolute and relative TTE, TTP, wash‐in, wash‐out, and maximum intensity were analyzed using general linear models followed by post hoc tests using Bonferroni's alpha correction procedure. The discrete data of the qualitative CEUS measurements, such as type of vascularization , presence of nonenhancing regions (yes or no), and origin of enhancement  were depicted in contingency tables and analyzed by using Fisher's exact test. Regarding the type of vascularization in pulmonary masses, sensitivity and specificity for detecting neoplastic transformation with the value rTTE were calculated. For all statistical tests, a Group seven contains rare masses in both the lung and mediastinum, which were found once and were therefore regarded as single values. No statistical analysis was done between group seven and the other groups. All statistical tests were selected and analyzed by a professional university statistician using IBM SPSS Statistics v24 .3n = 21), and one of each Abessiner, Burma, and Russian Blue. Dog breeds included mixed‐breed dogs (7), Golden Retriever (4), Boxer (2), Cavalier King Charles Spaniel (2), German Pinscher (2), and one of each of the following breeds: American Staffordshire Terrier, Bandog, Beagle, Bernese Mountain Dog, Cocker Spaniel, Collie, German Shepherd, German Shorthair, English Springer Spaniel, Flat Coated Retriever, Jack Russel Terrier, Labrador Retriever, Magyar Vizsla, Pug, Rottweiler, Shiba Inu, Shih Tzu, Tibet Terrier, and Berger Blanc Suisse.Sixty animals  met inclusion criteria for the study. Of the 24 cats, 13 were neutered males (54%) and 11 were spayed females (46%). Seventeen dogs were male  and 19 were female . The mean age of the feline patients was 11 years (range: 1–17 years), and that of the dogs was 10 years (range: 1–15 years). The mean weight in cats was 4.3 kg (range: 2–7 kg) and in dogs, 23 kg (range: 5–50 kg). The group of cats included Domestic Shorthairs  and histology in 19 of 60 cases (32%). Both procedures were applied in 13 of 60 cases (22%) and agreed in 11/13 cases (85%). In cases of disagreement, the histopathologic result was used. Of the 41 pulmonary masses, 14  were diagnosed as adenocarcinomas, seven  were undifferentiated carcinomas, nine  were sarcomas, one  lymphoma, and 10  were nonneoplastic mass lesions. The group of nonneoplastic masses included nine inflammatory consolidations and one lung lobe torsion.Of the 19 mediastinal masses seven  were diagnosed as thymomas, eight  as malignant lymphomas, and one of each of the following tumors: malignant nerve sheath tumor, hemangiosarcoma, tracheal B‐cell lymphoma, and melanoma.The patients were assigned into seven groups according to their cytology or histology results Table . Groups There was no significant difference regarding wash‐in, wash‐out time, TTP, and maximum intensity between the groups. In four patients, no contrast enhancement was observed. Two were adenocarcinomas, one was a lung lobe torsion, and one chronic pneumonia.P = 0.001; Figure P = 0.002).All continuous variables in the statistical analysis were normally distributed as assessed by a Shapiro‐Wilk test. The absolute TTE of the spleen represented systemic circulation time and differed significantly in both species (P = 0.002).The absolute TTE of the mass was compared to the absolute TTE of the spleen to calculate rTTE for each patient. Pulmonary arterial vascularized masses with an early rTTE had absolute TTE values of 1–12 s  in both species. In cats, TTE of these masses ranged from 1 to 12 s  and in dogs from 1 to 11 s . Bronchial arterial vascularized masses with a delayed rTTE had an absolute TTE of 1–28 seconds  in all patients . In pulmonary masses an early rTTE was observed in 16 cases and a delayed rTTE in 20 cases  thymomas during B‐mode ultrasonography, while four of seven 57%) thymomas showed nonenhancing areas with CEUS. Lymphomas enhanced uniformly in all cases , which was likely explained by the different cardiovascular status and circulation times in these species. Cats had a significantly earlier absolute TTE (mean 5.7 s) than dogs (mean 10.5 s). The values in dogs were slightly higher than previously reported in sedated canine patients.Enhancement in cats was significantly different from enhancement in dogs (Cats have a significantly shorter absolute TTE (mean 4.64 s) in intrathoracic mass lesions than dogs (mean 9.45 s) and an individual reference organ should be used in each patient.Five masses showed no contrast enhancement. With lung lobe torsion, the pulmonary blood flow is compromised and leads to varying contrast enhancement depending on the degree of torsion.In the present study, fine needle aspiration of the mass lesion was used to determine the final diagnosis and was performed in 90.1% of cases. Histology was carried out in 32% of the cases. Both modalities agreed in 11 of 13 (84.6%) cases, which is similar to previous studies in veterinary medicine with agreements of 82%.Enhancement should be compared to a systemically perfused organ where an actual hemodynamic situation is represented. Most of the patients were sedated with a small dose of butorphanol. The influence of anesthesia on CEUS has been studied and reports have shown that certain anesthetics have a significant influence on contrast enhancement in dogs and cats.The main limitation of this study was the relatively heterogeneous group of patients with different species and different cardiovascular conditions and the small numbers of dogs and cats within each subgroup. Use of the spleen as self‐reference were useful to classify enhancement patterns in each patient.All neoplastic lung masses were considered primary lung tumors. However, it cannot be completely ruled out that some of the masses diagnosed as primary lung tumors may represent metastases. Studies in human medicine do not indicate a significant difference between vascularization of primary and metastatic lung masses.In conclusion, CEUS of intrathoracic mass lesions provided useful information to characterize pulmonary and mediastinal mass lesions in this sample of dogs and cats. However, the test had low sensitivity and specificity for differentiating neoplastic pulmonary masses and therefore findings in individual clinical patients should be interpreted with caution.(a)Conception and Design: Hittmair KM(b)Acquisition of Data: Rick T, Hittmair KM, Schwendenwein I, Reifinger M(c)Analysis and Interpretation of Data: Rick T, Hittmair KM, Schwendenwein I, Ludewig E, Reifinger M(a)Drafting the Article: Rick T, Hittmair KM(b)Revising the Article for Intellectual Content: Hittmair KM, Kleiter M, Schwendenwein I, Ludewig E, Reifinger M(a)Final Approval of the Completed Article: Rick T, Hittmair KM, Kleiter M, Schwendenwein I, Ludewig E, Reifinger M


Dietary protein restriction did not alter emissions of contaminant gases nor microbial community structure in terms of diversity, although some genera were affected by the dietary challenge.To study the effect of dietary crude protein (CP) restriction in two different pig producing types and the role of gut microbiota, 32 pure castrated male Duroc and 32 entire male hybrid (F2) piglets were raised in a three-phase feeding regime with a restriction in CP content of the diets. The average body weight of hybrid animals were higher compared to Duroc pigs. No changes were found in average daily feed intake (ADFI) of hybrid animals in comparison to Duroc pigs. Hybrid animals apparently digested more CP than Duroc and Duroc pigs emitted more CH4 and ammonia emissions in Duroc pigs with respect to F2, no significant differences were found in contaminant gases emissions between diets. Moreover, LP diets did not alter the microbial community structure, in terms of diversity, although some genera were affected by the dietary challenge. Results suggest that the impact of reducing 2% of CP content was limited for reduction in contaminant gases emissions and highlight the hypothesis that moderate change in the dietary protein levels can be overcome by long-term adaptation of the gut microbiota. Overall, the influence of the producing type on performance and digestive microbiota composition was more pronounced than the dietary effect. However, both producing types responded differently to CP restriction. The use of fecal microbiota as biomarker for predicting feed efficiency has a great potential that should be completed with robust predictive models to achieve consistent and valid results.In order to reduce dietary nitrogen and achieve an efficient protein deposition as well as decrease N wastage, we challenged the nutrient utilization efficiency of two different producing types in front of a dietary crude protein (CP) restriction and studied the role of the microbiota in such an adaptation process. Therefore, 32 pure castrated male Duroc (DU) and 32 entire male hybrid (F2) piglets were raised in a three-phase feeding regime. At each phase, two iso caloric diets differing in CP content, also known as normal protein (NP) and low protein (LP), were fed to the animals. LP diets had a fixed restriction (2%) in CP content in regards to NP ones throughout the phases of the experiment. At the end of third phase, fecal samples were collected for microbiota analysis purposes and greenhouse gases emissions, together with ammonia, were tested. No changes were found in average daily feed intake (ADFI) of animals of two producing types (Duroc vs. F2) or those consumed different experimental diets (NP vs. LP) throughout the course of study. However, at the end of each experimental phase the average body weight (BW) of hybrid animals were higher compared to Duroc pigs, whereas a reverse trend was observed for average daily gain (ADG), where Duroc pigs showed greater values with respect to hybrid ones. Despite, greater CH Animals followed a three-phase feeding program . Diets (described in 2O3) diets underwent fecal spot sampling (≈50 g) by rectal stimulation at intervals of 8 h during 24 h. Representative fecal samples were stored at −20 °C until further digestibility analyses.Individual variations in body weight of the animals along with pen feed consumption were recorded weekly and individual average daily gain (ADG) was calculated as the slope from linear regression of the body weight on feeding days; the individual average daily feed intake (ADFI) was estimated from the weekly pen consumption, considering the number of animals in each pen. In addition, the efficiency of animals to convert feed into body mass was expressed as gain:feed ratio. At the last week of each feeding phase, those animals fed labeled  at the end of the third feeding phase (week 25 of age) was collected in a separate falcon tube (15 mL). These fecal samples were frozen instantly in liquid nitrogen, transferred to the laboratory, and stored at −80 °C.2, N2O, and CH4), together with NH3 emissions, were analyzed at the end of third experimental phase (week 25 of age) after the digestibility trial. A representative air sampling was obtained from the outside of the fattening installation and the midpoint of the exhaust air outlet in each module using the procedure suggested by Air Movement and Control Association (AMCA)  excreted and [Cr] intake are the concentration (ppm) of chromium in the feces and the feed, respectively.The coefficient of total track apparent digestibility CTTAD of nutrients was calculated using nutrient to marker ratio in the feed and fecal samples as follows:Fecal samples kept at −80 °C were freeze-dried and the DNA was extracted using a QIAamp DNA Stool Mini Kit  following the manufacturer’s instructions. The yield and purity of extracted DNA was assessed using a Nanodrop™ , by measuring the absorbance intensity at 260 nm and the absorbance ratio 260/280, respectively. Libraries were prepared using V3-V4 amplicons from the 16s rRNA gene. Sequencing was performed with Illumina Miseq , generating 902131 paired-end reads. Sequence data were analyzed following the UPARSE pipeline [The statistical analysis of the data was commensurate to the design of the study  considering pen as the experimental unit. Data were analyzed with SAS  using MIXED model procedure, assuming normal distribution of the data.i is the experimental phase , PTj is the producing type (Duroc and F2), Dik is the diet (LP and NP) along with their possible interactions, and Ɛijklmn is the error.Producing parameters  and coefficient of total tract of apparent digestibility of nutrients  data were analyzed as follows:3 emissions at the end of the third phase of study were analyzed as follows:i is the producing type (Duroc and F2), Dij is the diet (LP and NP) along with their possible interaction, and Ɛijk is the error.GHG data together with the NHp ≤ 0.05 and 0.05 < p ≤ 0.10, respectively.Tukey multiple comparison test was applied and significant differences and tendencies were declared at Biodiversity indices  and diets (LP and NP) were formulated based on recommendations published in FEDNA (2013) for animals of different ages. Normal and low CP diets were identical in covering the nutritional requirements of animals, except for CP where low CP diet was intentionally designed with less (≈2%) CP content compared to the normal one.p < 0.01) on all the studied parameters related with performance. No changes were found in ADFI of animals of two producing types (Duroc vs F2) or those that consumed different experimental diets (NP vs. LP) throughout the course of study (p > 0.05). In each phase, animals of two producing types were, on average, fed same amount of feed on the daily basis (p > 0.05); however, during the last phase, LP diet was consumed more than NP .A summary of performance parameters is provided in p = 1.0 for genotype and diet at the initial BW). However, at the end of each experimental phase, the average BW of F2 animals  were higher (p < 0.05) compared to Duroc pigs .At the entry of animals to the fattening facilities (9 weeks old) the piglets were distributed based on minimum BW variation to yield iso-weighed pens considering producing type (Duroc and F2) and diet (LP or NP). The data provided for the initial BW  show thep > 0.05). Thus, no variations in BW were found between animals fed different diets.Composition of diet (LP or NP) did not influence the BW throughout the feeding phases . Variations in performance led to a higher overall ADG in animals of F2 with respect to Duroc . Protein level in the diet did not influence the ADG of the animals throughout the experiment (p > 0.05). Neither producing type of the animals (Duroc and F2) nor CP content of the diet (LP and NP) influenced the efficiency of animals to convert feed into body mass expressed as gain:feed ratio (p > 0.05). No interactions were found between producing type of the animal and diet composition (PT × Di) in performance parameters measured during the experiment (p > 0.05).F2 animals showed numerically higher ADG with respect to Duroc ones in all the feeding phases, although differences did only reach statistical significance during the second (weeks 15 to 18 of age) phase . No differences were found in total tract digestibility of DM between Duroc and F2 or between animals fed NP or LP diets (p > 0.05). As the CP content recommended for each feeding phase reduced gradually in diets from first to third phase, the coefficient of total tract digestibility of CP decreased accordingly . Besides, in overall, animals of F2 genotype apparently digested more CP than Duroc , where no effect of diet was observed for the total tract digestibility of CP. The apparent digestibility of NDF was similar to that of CP and decreased (p < 0.01) along the phases of the experiment up to 21.6% at the third phase . No effects of producing type or CP content of the diet were observed in digestibility of NDF content (p > 0.05). Moreover, no interactions between main factors were found in CTTADs  measured during the experiment (p > 0.05).Results from CTTAD determination are showed in 4, CO2, and NO2) and ammonia (NH3), were monitored and the results are tabulated in p < 0.01) emitted  more CH4 (10.7 vs 5.6) and ammonia (3.3 vs 0.5) than F2 animals (p < 0.05). No differences were observed in NO2 and CO2 emissions either between diets or between producing types (p > 0.05).During the last  of the experiment, the emissions  of greenhouse gases  shared the 64.13% of the OTUs present in the analysis see  and onlyp < 0.05 in both cases).Richness index of diversity showed significant differences between genotypes and diets, with diversity being higher in NP diets and F2 animals (p > 0.05).Reads belonging to the kingdom Archaea accounted for an average of 0.15% of the total number of sequenced reads and no differences were found in the relative abundance of archaea between either producing types or diets  presented mean abundances above 0.1%. Firmicutes was the most abundant phyla in both genotypes (72.4 ± 6.13% in Duroc and 73.5 ± 4.81% in F2), followed by Bacteroidetes and Proteobacteria. Among main phyla, a genotype effect was found on Bacteroidetes, Proteobacteria, and Verrucomicrobia phyla , with Dup > 0.05), but in NP diets, Duroc animals presented a lower proportion of Firmicutes than F2 . Hence, the Bacteroidetes:Firmicutes ratio tended to be lower in Duroc animals  and Coriobacteriaceae (p = 0.004) from phylum Actinobacteria, family Prevotellaceae (p = 0.049) from phylum Bacteroidetes, and family Lachnospiraceae (p = 0.003) from phylum Firmicutes. On the other hand, F2 animals showed higher abundances in family Eggerthellaceae (p < 0.001) from phylum Actinobacteria; families Paludibacteraceae (p = 0.006), Porphyromonadaceae (p < 0.001), and Tannerellaceae (p < 0.001) from phylum Bacteroidetes; families Clostridiaceae (p = 0.003) and Peptococcaceae (p = 0.001) from phylum Firmicutes; and family Succinivibrionaceae (p = 0.036) from phylum Proteobacteria (A description of the main families identified within the four most abundant phyla is shown in bacteria .Geobacteraceae (p = 0.001) from phylum Proteobacteria, having higher abundances in animals fed NP diets than those fed LP diets. Interaction effects were present in families Clostridiales Family XIII, Incertae Sedis , and Eubacteriaceae  from phylum Firmicutes and family Geobacteraceae  from phylum Proteobacteria  and two related with differences between diets .SPLS-DA analysis enabled the selection of the most predictive or discriminative taxons in the data that helped to classify the samples according to either diet or genotype effect see ; from thp < 0.05, r > |0.58|) were considered. In Duroc animals, up to nine different genera and two phyla either positively or negatively were correlated with performance , meanwhile in F2 animals, this correlation was only found in two genera and one phylum. LP diets also presented more correlations between microbiota and performance (five genera and two phyla) than in NP diets (three genera and one phylum).We compared all the detected phyla and the most abundant bacterial genera , with those producing and digestive efficiency parameters studied in the trial  using SpConcern about the environmental pollution caused by the livestock sector has arisen exponentially and this concern involves greenhouse gases (mainly methane and carbon dioxide) emitted and nutrients wasted through the manure on the soil and water. In the present study, we tried to analyze the impact of a dietary challenge as CP reduction in EEAA-balanced diets on both fecal microbiota and environmental impact through pollutant gas emissions. Although we are aware of the use of balance studies to approach this issue, our main aim was to investigate the role of gut microbiota when adapting to CP restriction and their potential impact on the animal host.Experimental animals were kept under commercial conditions and they were raised in a three-phase feeding regime with the 2% restriction in LP diets that were fixed through the experiment. In agreement with the existing literature ,26,27,28LP diets did not alter the microbial community structure, in terms of diversity, although some genera were affected by the dietary challenge. These results are in agreement with a recent study , and sugBlautia sp. canine oral taxon 143 and Selenomonas bovis as the most influential taxa in the discrimination by diet. Blautia is a genus in the bacterial family Lachnospiraceae that phylogenetic analysis places within the Clostridium coccoides group, also referred to as the Clostridium Cluster XIVa [Blautia spp. may be to become a sink for reductive capability (H+) and so alternative pathway to methane synthesis [Selenomonas spp. is genus from phyla Firmicutes with a great activity as lactate producer; in both cases, the high proportion of cereals in the LP diet could have influence the proliferation of these types of bacteria.More than 95% of total sequences were shared across the animals used in this trial, which suggests a highly predominant and stable core population. SPLS-DA analysis showed ter XIVa . The comynthesis . SelenomThe authors are aware of the effect of castration on both parameters and the close interaction of breed and sex. However, in the real situation, breeds used to produce commercial male fatty pigs, are submitted to castration, whereas meat hybrids are not. Entire crossbreed animals are focused to produce low-price lean meat, whereas castrated fatty pigs (such as pure Duroc) are used to produce cured products with some specific features such as higher levels of intramuscular fat and precocity. The two extreme producing types allow us to study the animal’s resilience in a nutritional change  considering the key role of gut microbiota in such adaption processes. Effort has been done to study microbes’ interspecies interactions rather than titers and/or properties of singular microbes, considering that microbiota interactions may explain relevant aspects of gut functioning in swine, as has been demonstrated recently in human intestine ,34.p < 0.01) and ADG , although gain:feed ratio did not statistically differ between producing types (p = 0.57). Compared to other studies with a similar design [In this study, F2 animals had both higher body weight at slaughter ; however, the enteric methane emission was almost 2-fold in Duroc animals compared with F2 (p = 0.01). Such results are in agreement with the previous ones that indicate the methanogens abundance is not fully correlated with methane production, where certain key species may be responsible of a substantial part of the overall emission [In terms of microbiota profile and among all the diversity indices analyzed, Richness was the only one in which significant differences were found, where F2 animals had a higher number of OTUs in relation to Duroc animals . Hence, the Firmicutes: Bacteroidetes (F:B) ratio tended to be lower in Duroc animals  following a residuals linear regression, including factors such as producing type and initial weight of the animals see . In thisIn the present study, up to 14 genera and five phyla were found to be linked to any of the performance parameters analyzed . FibrobaPrevotella (negative correlation with CP digestibility in LP diets), Lactobacillus , and Ruminococcus  correlated with the performance parameters. Generally, data from Genera including To conclude, the influence of the producing type on pollutant gas emissions and fecal microbiota composition was more pronounced than the dietary CP quality effect, which lacked major significant influence. Both producing types appeared to respond differently to CP restriction and quality in terms of microbial composition and gas emissions, but further research should be conducted to confirm these preliminary results. The use of fecal microbiota as biomarker for predicting feed efficiency has a great potential, although it should be completed with robust predictive models.


Sclerotinia sclerotiorum, a total of 15 isolates of the Trichoderma species was screened for the biosynthesis of silver nanoparticles (AgNPs). Among them, the highest yield occurred in the synthesis of AgNPs using a cell-free aqueous filtrate of T.virens HZA14 producing gliotoxin. The synthetic AgNPs were charactered by SEM, EDS, TEM, XRD, and FTIR. Electron microscopy studies revealed that the size of AgNPs ranged from 5–50 nm and had spherical and oval shapes with smooth surfaces. Prepared AgNPs interacted with protein, carbohydrate and heterocyclic compound molecules, and especially, interaction patterns of AgNPs with the gliotoxin molecule were proposed. The antifungal activity assays demonstrated that percentage inhibition of the prepared AgNPs was 100, 93.8 and 100% against hyphal growth, sclerotial formation, and myceliogenic germination of sclerotia at a concentration of 200 μg/mL, respectively. The direct interaction between nanoparticles and fungal cells, including AgNPs’ contact, accumulation, lamellar fragment production and micropore or fissure formation on fungal cell walls, was revealed by SEM and EDS. These will extend our understanding of the mechanisms of AgNPs’ action for preventing diversified fungal disease.To control the disease caused by  Sclerotinia sclerotiorum (Lib.) de Bary is a significant plant pathogenic fungus that leads to sclerotium disease on many crops and causes economical substantial losses throughout the world  × 100, where I was the percentage inhibition of the radial growth of S. sclerotiorum hyphae, C was the colony diameter in the plate with sterile ddH2O and N was the colony diameter in the plate with AgNPs or 50% CFCL.To investigate the effect of AgNPs synthesized by ic waves . The serS. sclerotiorum, plates containing PDA were covered with the sterile cellophane membrane. A disc (3 mm) containing the 5-day-old hyphae of S. sclerotiorum was put into the center of a plate and incubated at 23 °C for 4 days and the cellophane membranes with colonies were transferred into the plates containing PDA with AgNPs of different concentrations , 50% CFCL and sterile ddH2O, as described above. The plates were incubated at 23 °C for 15 days. The number of sclerotia was counted on each plate and the percentage inhibition of produce using the same formula that was described above.For testing the effect of biosynthetic AgNPs on the sclerotial production of 2O. Plates were sealed by parafilm strips and incubated at 23 °C in dark conditions for five days. Sclerotia were surface-sterilized in 50% ethanol for two minutes, and dried on sterile filter paper under a laminar flow hood after rinsing with sterile water. Partial sclerotia were placed into the plate containing PDA and incubated at 23 °C for three days. Sclerotia producing hyphae were counted and the percentage of germination inhibition was evaluated using the same formula as described above.Antifungal effects of the synthetic AgNPs on sclerotial viability were also tested in vitro using the sterile soil . The 20 N 80, Oxford, UK) at 20 keV, as described above.To observe the interaction of AgNPs with fungal hyphae cells, sclerotia incubated for five and seven days were fixed in glutaraldehyde solution of 2.5% overnight for SEM observation, as described by . BrieflyT. virens HZA14 among 15 isolates , (200), (220), (311) and (222), respectively (The prepared AgNPs were characterized by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), transmission electron microscopy (TEM), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy analysis. The SEM micrographs showed that the external surfaces of spherical AgNPs synthesized by cell-free filtrate of e smooth a. The EDe smooth b, confire smooth c. The Agectively d, confirectively d. The un−1 and 3292 cm−1 were due to the bonded amine groups (–NH) or hydroxyl (–OH) in the interaction of fungal extract with AgNPs powder, respectively [−1 were attributed to asymmetric CH3, and symmetric and asymmetric CH2, stretching modes of carbohydrates and fatty acids. The peaks at 2360 and 2340 cm−1 were due to O=C=O stretching vibrations (CO2). The peaks at 1653, 1540 and 1077 cm−1 were attributed to the C=O (carboxylic group), C=C–C (aromatic ring or amide II group) and C–O or C–O–C stretching vibrations, respectively [−1 was assigned to the C–H bending vibration that was adjacent to the substituent group, indicative of heterocyclic compounds secreted by T. virens, which acted as the capping agent [−1. The peaks for the O–C and P–O–C groups in phospholipids, aromatics, amino acids (rocking vibrations) and ketones could be assigned near 568 cm−1 [−1 was attributed to the AgNPs binding with oxygen [FTIR measurements were carried out for identifying the functional groups involved in the AgNPs’ reduction . The broectively . The peaectively . A stronh oxygen ,33.−1 with a large shift change  revealed the AgNPs binding strongly with oxygen from the oxidized form of gliotoxin or negatively charged carboxyl groups in proteins. Secondly, the peak of the C–S–H group’s stretching vibrations, with remarkable shift change , was assigned at 668 cm−1 and suggested that the AgNPs were binding with sulphur from the reduced forms of gliotoxin [−1) of the peak at 832 cm−1 for the C–H bending vibration being adjacent to the substituent group. In addition, the peak of the R–CO–NH2 stretching vibrations near 1077 cm−1 also revealed the presence of proteins in synthetic AgNPs [Characteristics of FTIR spectra and the shift changes in AgNPs powder are presented in liotoxin . This coT. virens HZA14 against hyphal growth of S. sclerotiorum was assessed. The results showed that the AgNPs significantly inhibited the growth of fungal hyphae (p < 0.05), respectively .The inhibitory activity of AgNPs synthesized by the cell-free filtrate produced by l hyphae . The higectively . In addiS. sclerotiorum were covered onto the plates containing PDA with the AgNPs of different concentrations  for 15 days. The results showed that the synthesized AgNPs significantly reduced the number of sclerotia produced (2O).To determine inhibitory activity of AgNPs against sclerotial production, cellophane membranes (nine cm in dimeter) containing hyphae of produced . The perproduced . By compp < 0.05) (For determining the inhibitory activity of AgNPs against myceliogenic germination of sclerotia (MGS), 20 mL AgNPs suspension with different concentrations  were sprayed on sclerotia on the surface of the soil and treated at 23 °C for five days. The results of myceliogenic germination of sclerotia on PDA indicated that the MGS was completely inhibited after treatment with three different concentrations of AgNPs  . The 80% < 0.05) . ComparaTo observe the interaction of the AgNPs with hyphae cells, morphological changes on the surfaces of sclerotia were seen by SEM after sclerotia were treated at a concentration of 200 μg/mL AgNPs for five or seven days. The micrographs of SEM showed that the small lamellar fragments a or micrAgNPs have unique optical, electrical, and thermal properties, which constitute the basis of novel applications in antimicrobial, anticancer, larvicidal, catalytic, and wound healing activities . They arT. virens HZA14, showing great difference among isolates of different species  of the sulphur element was higher than that (1.04%) of the nitrogen element. FTIR study revealed the presence and binding of protein, carbohydrates and heterocyclic compounds and fatty acids with AgNPs. It is well known that the proteins bind with nanoparticles through cysteine residues or free amino groups [T. virens [T. longibrachiatum [The synthetic yield was involved in components of secondary metabolites secreted by eviously . Characto groups . Electroo groups . However. virens , the intachiatum , synthesS. sclerotium, showing their potential applications against white mold, which is partially similar to a previous report [The AgNPs exhibit their antimicrobial potential through multifaceted mechanisms . It is is report . Howevers report ,43. Howes report . Whethers report , for achT. virens HZA14 producing gliotoxin against the soilborne pathogen S. sclerotiorum in vitro. Characterization of AgNPs revealed that the highest yield was related to gliotoxin produced by T. virens HZA14, and meanwhile, interaction patterns of AgNPs with gliotoxin molecules were proposed. The biosynthesized AgNPs showed a high percentage inhibition against hyphal growth, sclerotial formation, and myceliogenic germination of sclerotia. All the inhibitory behaviors observed using SEM/EDS technologies may be mostly attributed to nanoparticle–cell direct contact, accumulation, lamellar fragment production and micropore or fissure formation on fungal cell walls and may be related to the alteration of fungal cell walls, while these changes provide the channels for the AgNPs to enter the cells. The final cell death may be associated with cell membrane damage, oxidative stress, and altered transport activity and signal transduction pathways.In conclusion, this study demonstrated for the first time the prominent antifungal activity of AgNPs synthesized using a cell-free aqueous filtrate of


For the Moderna vaccine mRNA-1273, the appraisal shows: relative risk reduction, 94.1%; 95% CI, 89.1% to 96.8%; p = 0.004; absolute risk reduction, 1.1%; 95% CI, 0.97% to 1.32%; p < 0.000. Unreported absolute risk reduction measures of 0.7% and 1.1% for the Pfzier/BioNTech and Moderna vaccines, respectively, are very much lower than the reported relative risk reduction measures. Reporting absolute risk reduction measures is essential to prevent outcome reporting bias in evaluation of COVID-19 vaccine efficacy.Relative risk reduction and absolute risk reduction measures in the evaluation of clinical trial data are poorly understood by health professionals and the public. The absence of reported absolute risk reduction in COVID-19 vaccine clinical trials can lead to outcome reporting bias that affects the interpretation of vaccine efficacy. The present article uses clinical epidemiologic tools to critically appraise reports of efficacy in Pfzier/BioNTech and Moderna COVID-19 mRNA vaccine clinical trials. Based on data reported by the manufacturer for Pfzier/BioNTech vaccine BNT162b2, this critical appraisal shows: relative risk reduction, 95.1%; 95% CI, 90.0% to 97.6%;  Using messenger RNA (mRNA) in vaccines to produce proteins that trigger an immune response against infectious diseases has held promise for decades, but until recently, no clinically tested mRNA vaccine has managed to advance beyond small, early-phase trials . NormallHowever, several biological obstacles continue to challenge the development of mRNA vaccines, including “mRNA’s extremely large size, charge, intrinsic instability, and high susceptibility to enzymatic degradation” . To mitiBMJ Opinion, 26 November 2020 [As noted in ber 2020 ,“There may be much more complexity to the ‘95% effective’ announcement than meets the eye—or perhaps not. Only full transparency and rigorous scrutiny of the data will allow for informed decision making. The data must be made public.”BMJ Opinion, Pfizer/BioNTech and Moderna reported the relative risk reduction of their vaccines, but the manufacturers did not report a corresponding absolute risk reduction, which “appears to be less than 1%” [p = 0.000, and 1.1%; 95% CI, 0.97% to 1.32%; p = 0.000. The same publicly available data, without absolute risk reduction measures, were reviewed and approved by the roster of members serving on the U.S. Food and Drug Administration’s (FDA’s) Vaccines and Related Biological Products Advisory Committee (VRBPAC) for emergency use authorization (EUA) of the mRNA vaccines [As was also noted in the than 1%” . Absolutthan 1%”  and Modethan 1%”  are, resvaccines . Ironicavaccines . The FDA“Provide absolute risks, not just relative risks. Patients are unduly influenced when risk information is presented using a relative risk approach; this can result in suboptimal decisions. Thus, an absolute risk format should be used.”New England Journal of Medicine also published clinical trial data on safety and efficacy for the BNT162b2 vaccine [The  vaccine  and the  vaccine , but witThe present article uses epidemiologic tools to critically appraise absolute and relative risk reduction measures for vaccine efficacy in phase III clinical trials of the COVID-19 mRNA vaccines. Microsoft Excel was used to analyze data and chart risk reduction outcomes. The article further clarifies how selective reporting of vaccine efficacy measures can cause a type of outcome reporting bias that misrepresents health information disseminated to the public.The application of epidemiologic and biometric methods to clinical diagnosis and treatment is known as clinical epidemiology . Clinican, is represented by a + b for the vaccine group and c + d for the placebo group.The following 2 × 2 contingency tables for SARS-CoV-2 infection are based on reported clinical trial data for the Pfzier/BioNTech BNT162b2 vaccine  and the The following epidemiologic equations use data from the 2 × 2 contingency tables  to calcuRisk ratio (RR):a/(a + b), by the control event rate (CER), c/(c + d) [The risk ratio, also known as the relative risk, in a randomized controlled trial is the ratio calculated by dividing the experimental event rate (EER), /(c + d) . DividinRisk ratio 95% confidence interval (CI):The risk ratio 95% confidence interval predicts the range of probable risk ratios if the experiment or trial was repeated 95 out of 100 times. The narrower the range between the upper and lower CI values, the more precise the CI. If the range includes the RR null value, 1, the risk ratio is considered statistically insignificant. The equation calculates the standard error (SE) ,21, and Absolute risk reduction (ARR):The absolute risk reduction is a percentage equal to the arithmetic difference when subtracting the EER from the CER . The difAbsolute risk reduction 95% confidence interval :The standard error in the absolute risk reduction 95% confidence interval measures the square root of the sum of the group variances . If the Number needed to vaccinate (NNV):The NNV, or the number needed to vaccinate to prevent one infection, is the reciprocal of the ARR . Note thNNV 95% confidence interval (CI):The CI of the NNV is calculated by dividing 1 by the ARR CI , again mRelative risk reduction (RRR) or vaccine efficacy (VE):The relative risk reduction is the same as vaccine efficacy (VE) . The RRRRRR, VE 95% confidence interval (CI):The CI for the relative risk reduction is calculated by subtracting the RR CI from the null value 1.Pvalues, which measure the probability that a trial result occurred by chance, can be calculated from the confidence interval for the difference between two proportions, as in the ARR, and from the confidence interval for a ratio, as in the RRR . Online Clinical epidemiologic tools can be used to critically appraise the efficacy of new COVID-19 vaccines having biological mechanisms that differ from the mRNA vaccines, such as AstraZeneca-Oxford’s ChAdOx1 adenoviral vector vaccine  and JohnMedical and public health experts continue to stress the need to include measurements of absolute risk reduction and number needed to treat when reporting results of clinical interventions . Current“…critical appraisal knowledge and skills are limited among physicians,” and “use of relative effect measures was associated with greater perceptions of medication effectiveness and intent to prescribe, compared with the use of absolute effect measures.”Reporting relative measures may be sufficient to summarize evidence of a study for comparisons with other studies, but absolute measures are also necessary for applying study findings to specific clinical or public health circumstances . OmittinSimilar to the critical appraisal in the present article, critical appraisals of reported vaccine efficacy in other studies reveals clinically significant insights. For example, a 2018 review of 52 randomized trials for influenza vaccines that studied over 80,000 healthy adults reported an overall influenza vaccine EER of 0.9% and a 2.3% CER, which calculates to a RRR of 60.8% . This vaThe mRNA vaccine manufacturers reported that infections in most subgroups in phase III clinical trials were similar for both vaccines after two doses. Vaccine clinical trial case definitions for SARS-CoV-2 infection included COVID-19 clinical symptoms; thus the trials were not designed to provide evidence of vaccine efficacy for protection against asymptomatic infections. In addition to outcome reporting bias, information bias may have also affected COVID-19 vaccine trial outcomes due to misclassification of SARS-CoV-2 infections as mild adverse effects of the vaccines. For example, several COVID-19 clinical symptoms are similar to the vaccines’ adverse effects such as fever, pain, and fatigue, which could potentially lead to missed diagnoses of viral infections.A limitation of this article is that it only critically appraised mRNA vaccine efficacy in healthy individuals who were randomized to two groups under strictly controlled conditions. The critical appraisal did not include vaccine safety and effectiveness outcomes within a general population that includes unhealthy people and that lacks control over confounding factors. For example, healthy vaccinee bias occurs when people who are in better health are more likely to follow vaccination recommendations in order to protect their health .A critical appraisal of phase III clinical trial data for the Pfizer/BioNTech vaccine BNT162b2 and Moderna vaccine mRNA-1273 shows that absolute risk reduction measures are very much lower than the reported relative risk reduction measures. Yet, the manufacturers failed to report absolute risk reduction measures in publicly released documents. As well, the U.S FDA Advisory Committee (VRBPAC) did not follow FDA published guidelines for communicating risks and benefits to the public, and the committee failed to report absolute risk reduction measures in authorizing the BNT162b2 and mRNA-1273 vaccines for emergency use. Such examples of outcome reporting bias mislead and distort the public’s interpretation of COVID-19 mRNA vaccine efficacy and violate the ethical and legal obligations of informed consent.


Bothrops snakes, but only a few studies have attempted to identify the targeted paralogues or the molecular mechanisms involved in modifications of gene expression during ontogeny. In this study, we decoded B. jararacussu venom gland transcripts from six specimens of varying sizes and analyzed the variability in the composition of independent venom proteomes from 19 individuals. We identified 125 distinct putative toxin transcripts, and of these, 73 were detected in venom proteomes and only 10 were involved in the ontogenetic changes. Ontogenetic variability was linearly related to snake size and did not correspond to the maturation of the reproductive stage. Changes in the transcriptome were highly predictive of changes in the venom proteome. The basic myotoxic phospholipases A2 (PLA2s) were the most abundant components in larger snakes, while in venoms from smaller snakes, PIII-class SVMPs were the major components. The snake venom metalloproteinases (SVMPs) identified corresponded to novel sequences and conferred higher pro-coagulant and hemorrhagic functions to the venom of small snakes. The mechanisms modulating venom variability are predominantly related to transcriptional events and may consist of an advantage of higher hematotoxicity and more efficient predatory function in the venom from small snakes.Ontogenetic changes in venom composition have been described in  Advanced snakes (clade Caenophidia) have acquired the ability to produce, secrete, and inject venoms. The evolution of lethal toxins and the appearance of the sophisticated injection system provided snakes a great advantage in the evolutionary process to subdue prey . Snake v2 (PLA2), and C-type lectins (CTLs)  benzoic acid  incubated for 40 min at 37 °C and activity determined according to the absorbance at 425 nm and expressed as Absorbance/min/μg of venom. SVMPs and PLA our lab . For SVMThe coagulant activity of individual venoms was assessed in citrated human plasma from healthy volunteers under the approval of the Ethical Committee (CAAE: 89499218.8.0000.5377). Participants read and signed the written informed consent form before enrollment. Different concentrations of venom samples were diluted to in 25 μL of phosphate-buffered saline (PBS), incubated with 100 μL of plasma at 37 °C, and the clotting time measured in a coagulometer . The coagulant activity was expressed as Minimum Coagulant Dose (MCD), which corresponds to the amount of venom that induces clotting of human plasma in 60 s.2 chamber, the dorsal skin was removed, and the hemorrhagic halos were measured. Groups of 3 animals were tested, and control group animals were injected with PBS only. The myotoxic activity was assayed using 50 µg of venom pools in 30 µL of PBS, which was injected intramuscularly into the gastrocnemius muscle in Swiss mice. After 3 h, the animals were bled via ophthalmic plexus, and the sera were assayed for creatine–kinase activity with a commercial kit CK-UV , according to the manufacturer’s instructions. Groups of 4 animals were tested, and animals from the control groups were injected with PBS only. To determine the lethal activity as described by Moretto Del-Rei et al. [The amount of venom extracted from each snake was not sufficient to perform in vivo experiments individually. Thus, the venoms were grouped in 3 pools according to snake size: Pool 1 comprised venoms from smaller individuals , pool 2 included a group of intermediate individuals , and pool 3 grouped venoms of larger individuals . Hemorrhagic, myotoxic, and lethal activities were carried out using male Swiss mice (18–20 g) kept under controlled temperature and light periods with water and feed ad libitum, under the approval of the Butantan Institute Ethics Committee on Animal Use (Protocol Number: 5091020819). Briefly, for hemorrhagic activity, samples containing 10 µg of each venom pool, diluted in 50 µL of PBS, were injected intradermally into the dorsal skin of mice. After 3 h, the animals were euthanized in a COi et al. , samplest-test  or One-Way ANOVA using the software GraphPad Prism . Differences between groups were considered statistically significant at * p < 0.05; ** p < 0.01 and *** p < 0.001.The statistical analysis was performed using the software GraphPad Prism . The normality of the data was assessed using the D’Agostino–Pearson normality test and normal probability plots. Data were log-transformed when necessary to meet the assumptions of parametric statistical tests. We used a correlation analysis (Pearson and Spearman correlation) and multiple linear regression to verify the association between the SVL, sex, locality of collection, reproductive status, and expression data of the transcripts or/and enzymatic activities. Differences between two groups were analyzed by the Student’s