Inga seedlings in two different but neighboring forest ecosystems in the undisturbed tropical Amazon rainforest at the Tiputini Biodiversity Station in Ecuador. In terra firme plots, which were situated higher up and therefore typically outside of the influence of river floods, and in várzea plots, the lower part of the forest located near the riverbanks and therefore seasonally flooded, tree seedlings of the genus Inga were randomly collected and measured, and the rhizosphere soils surrounding the root systems was collected. Members of the Fabaceae family and the genus Inga were highly abundant in both forest ecosystems. Inga sp. seedlings collected in terra firme showed a lower shoot to root ratio compared to seedlings that were collected in várzea, suggesting that Inga seedlings which germinated in várzea soils could invest more resources in vegetative growth with shorter roots. Results of the physical-chemical properties of soil samples indicated higher proportions of N, Mo, and V in terra firme soils, whereas várzea soils present higher concentrations of all other macro- and micronutrients, which confirmed the nutrient deposition effect of seasonal flooding by the nearby river. ITS metabarcoding was used to explore the mycobiome associated with roots of the genus Inga. Bioinformatic analysis was performed using Qiime 2 to calculate the alpha and beta diversity, species taxonomy and the differential abundance of fungi and arbuscular mycorrhizal fungi. The fungal community represented 75% of the total ITS ASVs, and although present in all samples, the subphylum Glomeromycotina represented 1.42% of all ITS ASVs with annotations to 13 distinct families, including Glomeraceae , Gigasporaceae , Acaulosporaceae . AMF spores of these three AMF families were morphologically identified by microscopy. Results of this study indicate that AMF surround the rhizosphere of Inga seedlings in relatively low proportions compared to other fungal groups but present in both terra firme and várzea Neotropical ecosystems.This study explored the composition of the mycobiome in the rhizosphere of  The Amazon is estimated to comprise 50% of the remaining global tropical forests on our planet and is considered a biodiversity hotspot with endemic and endangered flora and fauna , a pristine and undisturbed tropical research area situated in the Yasuní Biosphere Reserve in the Orellana Province of Ecuador. We measured the roots and shoots of Inga seedlings and collected rhizosphere soil samples to measure soil physical and chemical properties, and to extract DNA to perform metabarcoding methods to assess what fungi, with a special focus on AMF, were present in the rhizosphere of Inga sp. Moreover, we were interested whether the abundance and composition of the Inga rhizosphere mycobiome differed between terra firme and várzea sampling sites, and what factors could influence these possible differences. Furthermore, we used microscopy to morphologically identify AMF spores to compare them with the metabarcoding results. Considering the differences in forest structure and abiotic factors within each ecosystem, we expected to find AMF to be present in both ecosystems, but the AMF community structure to differ in response to these conditions. Hence, in terra firme forests, where soils tend to be poorer in nutrients in comparison to várzea soils, a higher degree of AMF colonization is expected, and possibly a higher abundance and diversity of AMF in close proximity to the roots of Inga seedlings.The present study investigated the fungal abundance and diversity in terra firme and várzea soils associated to the roots of tree seedlings of the genus nd fungi . Members22.12, occupies a unique location at the intersection of the Andes and the Amazon. Yasuní’s climate is characterized by relatively high temperatures , and a high relative humidity of 80–94% throughout the year (The current study took place at the Tiputini Biodiversity Station (TBS), situated within a pristine area of primary Amazon rainforest with minimum human impacts in the province of Orellana, Ecuador Figure 1the year . Meteorothe year . The TBS2.2Inga of várzea and terra firme forests were sampled. Sampling collection details are shown in Inga seedlings was observed, particularly in areas with visual evidence of recent flooding. Areas closer to the Tiputini river presented distinct physical and biological structures related to the influence of seasonal floods, such as the presence of aerial roots, general suppression of plant growth and the formation of erosion slides. Within established TBS 1 ha plots, the total number of subadult and adult trees in terra firme plots  and várzea  have been assessed from 1998 until present. These numbers were used to calculate local relative abundances on the family (Fabaceae) and genus (Inga) level within terra firme and várzea forest plots. The total abundance of Fabaceae trees within terra firme plots of the TBS was 13.2%, with almost half of these trees belonging to the genus Inga (5.7%). Within várzea forests, 25.4% of all subadult and adult trees encountered in the vegetation belonged to the Fabaceae family, of which more of half of the individuals (14.2%) could be identified as Inga sp. . Although the distribution of seedlings of Inga sp. was not homogenous along the station trails, in general it was not difficult to encounter them.Within the TBS research station, during three fieldwork expeditions between November 2018 and February 2020, tree seedlings of the genus 2.3Inga sp. seedlings in várzea and terra firme forests. During sampling, Inga sp. seedlings where excavated, and soil clumps attached to the seedling root structure were collected for physical and chemical soil analysis. To take a more representative sample soil, it was collected from varying depths, between 5 cm to 30 cm from the soil surface. 250g of soil was stored in 50 ml Falcon tubes. Soil samples were transported to the USFQ campus and stored at 4 °C. Samples where dried and sieved before processing at the laboratory facilities of the USFQ Environmental Engineering Department. Soil samples were characterized using the EPA 3050B method for initial sample mineralization and EPA 6010B for instrumental measurement. Soil analysis was conducted using a plasma spectrophotometer  with inductive coupling and optical detector (ICP-OES). Certipur® standards were used for instrument calibration, with an ICP multi-element standard solution VIII . For quality control in recoveries, estuarine sediment as the Standard Reference Material (SRM®) from NIST® number 1646 was used .Soil samples were collected from the rhizosphere of 2.4Inga genus is characterized by the presence of winged rachis, a morphological characteristic that permits easy identification in the field  during two sampling expeditions in November 2018 and August 2019. Soil aggregates attached to the roots system were collected and stored in 1.5 ml Eppendorf tubes with LifeGuard Soil Preservation solution. Rhizosphere samples were transported to USFQ and stored at -20°C. The total DNA of the rhizosphere samples (< 2g of soil) was extracted by lysis with mechanical spheres. DNA purification was performed using the DNeasy PowerSoil kit following the manufacturer’s instructions . Extracted rhizosphere DNA samples were shipped to Baseclear  for metabarcoding using the Internal Transcribed Spacer (regions ITS1 and ITS2) as a molecular marker , a representative sequences table, and a statistical table . The sampling depth for Fungi ASVs was 10000 and 96 for Glomeromycotina. Alpha diversity was calculated using Shannon’s diversity index, Observed Features, Faith’s Phylogenetic Diversity and Evenness. Statistical tests were performed and we used the Kruskal-Wallis test , the terminology used to describe subcellular spore wall structure is based on the spore development model as proposed by https://invam.ku.edu/). Nomenclature for AMF genera and classification follows Spores were cleaned and collected manually using an extruded 9-inch glass pipette, and separated by morphotypes based on spore color, size, and type of spore formation  and mounted in polyvinyl alcohol lacto glycerol (PVLG)  and PVLG2.7Inga tree seedlings, a principal component analysis (PCoA) was performed to construct a 2D graph to visualize correlations between the relative abundance information of the taxonomic composition of fungal ASVs and all chemical properties measured as previously described. The larger the distance between two coordinates in the PCoA graph, the less similar they are. We used the ‘envfit’ function of the vegan R package to calculate the environmental vectors, and the PCoA plot was created using the ‘ggplot2’ package (R version 4.2.2).To display the differences in the fungal ASV composition as present in 3 terra firme and 3 várzea samples as collected from the rhizosphere of 33.1-06, p=0.05), presented the highest statistical difference for all elements measured and was found at considerably higher concentrations for soil samples collected in várzea, than in terra firme. In contrast, vanadium (V) (t(8)=0.026, p=0.05), presented higher concentration in terra firme forests than in várzea forests.During each field expedition, sample site descriptions were conducted to evaluate visible soil characteristics, and to assess possible differences between sample sites. It was observed that in the terra firme plots, leaf litter and humus accumulation was visibly higher, less compacted, and less degraded. In várzea forest sites, leaf litter and humus accumulation in these soils had a darker colour, tended to be waterlogged, and was more compressed into a thinner layer. Macro- and micronutrients with important roles in plant development were evaluated for soil samples collected at várzea and terra firme sites at the TBS, and presented a normal distribution, which was confirmed using a Kolmogorov-Smirnov test  than those collected in terra firme sites (10.66 cm ± 3.7 cm). On the other hand, shoots of Inga seedlings collected in várzea plots had the tendency to be longer (23.54 cm ± 7.41 cm), than those sampled in terra firme plots (17.73 cm ± 3.99 cm). To further explore the relationship between shoot and root lengths between ecosystems, a shoot and root ratio was calculated  and terra firme (n=10) forest ecosystems. Ten samples were collected in November 2018, and 9 samples in August 2019. The ITS metabarcoding results showed a total of 1 207 868 raw sequences (Paired End Sequences), with 5776 identified amplicon sequence variants (ASVs) using DADA2. The median of the quality score of the data was 30. Five samples were excluded from the analysis, due to the low numbers of features, which established 8 rhizosphere samples from terra firme, and 6 samples from várzea ecosystems. Taxonomic analysis was performed to identify the rhizosphere mycobiome composition, with ITS ASVs annotate to 10 kingdoms, of which Fungi was dominant , followed by an unidentified kingdom , Viridiplantae , Alveolata . The remaining 0,8% corresponded to other minor kingdoms . For the Fungi kingdom, evenness and Shannon indexes across collection dates were significantly different . However, the richness across ecosystems was not significantly different . Finally, the α-diversity within the identified Glomeromycotina did not reveal clear composition differences neither between ecosystems nor collection dates . Moreover, the rhizosphere composition indicated a collection date effect, as both Bray Curtis and Weighted UNIFRAC PERMANOVA tests showed significant differences between samples collected in November 2018 compared to samples collected in August 2019 . Similar trends were observed analyzing only Fungi ASVs, where Jaccard and Unweighted UNIFRAC revealed significant differences between ecosystem . Finally, the β-diversity calculations for ASVs annotated to the phylum Glomeromycota (now subphylum Glomeromycotina) did not show differences for sampling location nor sampling date . We used GNEISS metrics to calculate and identify differentially abundant fungal taxa by comparing ecosystems (terra firme vs várzea) and sampling date (November 2018 vs August 2019). When analyzing exclusively ASVs belonging to the kingdom Fungi, the most abundant taxon in várzea samples was annotated to the Sordariomycetes class, while in the terra firme samples the most abundant taxons were annotated to the genus Scedosporium and two members of the Nectriaceae family . A factor that could explain the high abundance of this group might be their ability to form rhizospheric associations with both nitrogen fixing bacteria and AMF. These plant-associated microorganisms promote plant health by positively stimulating plant growth, boosting physiological responses towards biotic and abiotic stress factors, and supporting the plant’s adaptation to environmental circumstances  can be found below: The study was designed by AH-D, PH, and JD. Research permits were managed by SZ and PH. Samples were taken by AH-D, PH, and JD. AH-D and PH prepared the samples for physical chemical soil analysis and amplicon sequencing. VA-G and BP-V conducted the bioinformatics analyses. JD prepared the soil, root, and spore samples and executed the mycorrhizal potential experiments. PH and JD complemented the analyses with their specific expertise and interpreted the overall data. AH-D, PH, JD, VA-G, and BP-V visualized the results and wrote the manuscript. All authors read and approved the final version of the manuscript.


P ≤ 0.05) by ZnO-NPs supplementation. Zinc methionine supplementation significantly improved liver function and decreased seminal plasma TBARs. Treatments with Zn-Met and ZnO-NPs increased seminal TAC and blood testosterone levels with reduced blood cortisol levels compared to other groups. Severe heat stress could be counteract by inclusion Zn with studied forms Zn-Met and ZnO-NPs at recommended dose (30 mg/kg diet) to improve semen quality and antioxidant status.The harmful influences of global warming on rabbit reproduction and industry attract global attention. Zinc (Zn) is an important trace element with a wide list of functions in the male reproductive system. The aim of this study was to estimate the effects of different forms of zinc supplementation, as organic (Zn methionine), nano (nano Zn oxide) as indirect way to minimized it impact on environment, and inorganic (Zn sulphate) on physiological parameters, semen quality, anti-oxidative status, hormonal profiles of male rabbits subjected to server heat stress. Thirty-six V-line bucks  were randomly distributed to 4 groups, bucks in 1st group  fed basal diet without Zn supplementation, the 2nd, 3rd and 4th groups fed basial diet with 30 mg/kg of zinc methionine (Zn-Met), nano zinc oxide (ZnO-NPs), and zinc sulphate (Zn-S), respectively for a period of 10 weeks suffered from severe heat stress of environmental ambient temperature . Semen samples were collected and evaluated for volume, pH, motility, concentration, viability, initial fructose, and seminal plasma antioxidant concentration. Liver, kidney function, hormonal and antioxidant profile were measured in blood serum. The results revealed that, compared to control, all forms of Zn supplementation used significantly improved kidney function (creatinine), serum antioxidant (SOD and CAT), physiological parameters, especially on 1st month of the experiment, and seminal plasma antioxidant (SOD and CAT) of heat stressed bucks. Likewise, semen quality in terms of sperm concentration, sperm viability, and initial fructose enhanced significantly ( Moreover, many blood biochemicals dis-balances in male rabbit were occurred by exposure to thermal stress2. Sperm are rich in polyunsaturated fatty acids with low levels within the cytoplasm3. Heat stress caused a higher level of inflammatory responses in the blood, resulting in a decrease in immunological function and a potential for apoptosis spermatozoa4. In addition, thermal stress caused an increase in oxidative stress, which in turn increased tissue damage by accelerating lipid peroxidation2 and significantly weakening antioxidant defense5, with lipid peroxidation followed in cellular damage6.Recently, global warming and heat stress represent the main challenges that contribute to the significant reduction in rabbit semen quality, leading to a significant decrease in sexual behavior, hormonal imbalances, an increase in abnormal sperm; a drop in sperm production, and finally a decrease in fertilization capacity and fertility7. Many microelements are successfully used to reduce the damaging effects or avoid harmful impacts of heat stress1. Zinc is one of many series components that control the oxidative stress of cells8. In addition, zinc is the second richest trace element after iron, which plays an important role in the male reproductive process because of their high activity at the molecular level10, and involved in various enzyme activities and hormone functions11. Zinc is an effective antioxidant owing key role to catalytic, coactive, or structural roles in different cofactor including more than 300 enzymes12 such as SOD; more than 2000 transcription factors13, involved in the biosynthesis of DNA and cell division14, gene expression15, and immune protection16. Overall, zinc acts as an antioxidant stress agent by inhibition of oxidation of macromolecules such as DNA and proteins, as well as the inhibition of inflammatory response, subsequently downregulation of ROS production17. Inorganic zinc bioavailability in animals is low and large amounts of zinc are excreted into faeces, resulting in environmental pollution and waste of zinc resources. Nano-zinc can be used at lower doses and indirectly prevents environmental contamination18. The nano-zinc (particle size is below 100 nm) is more effective, available, and absorbed easier19, while it bypasses through the stomach wall and into body cells more quickly than ordinary minerals with larger particle size20.Several methods have been used to prevent the negative impacts of heat stress in buck rabbits, including phytogenic, minerals, and vitaminsTherefore, the objective of the present study was to determine whether the use of various zinc sources, such as zinc methionine, nano zinc oxide and zinc sulphate, under heat stress conditions could improve the semen of the quality of the rabbit bucks, their hormonal status, and their antioxidant status.The study was carried out at the Department of Animal and Fish Production, Faculty of Agriculture (Saba- Basha), Alexandria University, Alexandria, Egypt . All experimental procedures and handling of buck rabbits were approved by the Alexandria University Institutional Animal Care and Use Committee , during the summer season (July to September 2021). The experiment was carried out according to relevant guidelines and regulations. Our animal research reports follow the recommendations of the ARRIVE Guidelines.® Zn was purchased from United BioMed for feed additives, Cairo, Egypt), nano zinc oxide  and zinc sulphate , respectively according to recommendation doses of22 for ZnO NPs and close to recommendation doses of23 for Zn-Met and ZnS. The experiment was carried out according to relevant guidelines and regulations. The experiment lasted 10 weeks. Feed and fresh clean water were provided ad libitum. The basic diet was formulated as  and the calculated chemical composition was  according to the recommendation of24.Thirty-six fertile V-line male rabbits, 6–7 months old with body weight (2842.29 ± 34.46 g), were used. Live body weights of rabbit bucks were recorded at the beginning of the experiment and at the end. Body weight change was calculated. The bucks were individually caged in wire batteries with a normal daylight (16–17 h). All rabbits were adapted in the experimental rabbit shed for 2 weeks. The rabbits were randomly distributed into four groups (n = 9 bucks per group) as follows: the first group (control group) fed a base diet without Zn supplementation, the second, third and fourth groups fed a base diet with 30 mg/kg of zinc methionine , ambient temperature (°C) and relative humidity (%) were recorded daily at midday using a Mercury-room thermometer and hygrometer, respectively. the weekly temperature-humidity index (THI) was determined by the equation defined byThe rectal temperature (RT) and the respiration rate (RR) were recorded twice (4th and 8th weeks of the experiment) at 12:00–2:00 PM. A clinical digital thermometer and stopwatch were used to measure RT by inserting the thermometer 2 cm into the rectum for 2 min, inclined toward the wall of the rectum. Visual counting of nasal movement per 1 min without disturbing the rabbits by using a hand counter to determine the RR.27, giving an arbitrary score of 0 to 3. The individual progressive motility was examined by diluted semen with normal saline (1:100) and transferred to a warm glass slide (37 °C) covered with a cover slip and examined under a light microscope (400× magnification) on a scale of 0 to 100% according to28. Sperm concentration (*106/mm3) was evaluated in duplicate using a Neubauer hemocytometer after dilution with distilled water (1:200). Sperm viability was determined using semen smears mixed with Eosin-Nigrosin stain and viable and non-viable spermatozoa were determined by counting 200 sperm under a light microscope29. The initial fructose concentration of the collected semen was determined by a spectrophotometer using commercial kits  according to the manufacturer's instructions. At the 10th week of the experiment, the seminal plasma of 4 bucks of each treatment was separated from the mass of the sperm cells by centrifugation at 700g for 15 min. The supernatant  was collected and kept at − 20 °C until analysis. Seminal plasma SOD, CAT, TBARs, and TAC were verified by colorimetric techniques using commercial kits , according to the manufacturers’ instructions.The semen samples of each rabbit buck were collected weekly in the morning (8 am) for 10 weeks using an artificial vagina by exposing a mature female rabbit to the bucks as a teaser during collecting. After collection, the ejaculate volume without gel was recorded in graduated collecting tubes to the nearest 0.01 ml (excluding the gel plug). Semen pH was directly measured by using pH paper strips . Sperm mass activity was determined manually according tog for 10 min to obtain serum. The serum obtained was transferred to sterile Eppendorf® tubes and stored at − 20 °C. Serum samples were analyzed for AST and ALT according to30, urea31, creatinine33, SOD34, CAT36, TBARS37, and TAC38. These analyses were performed using commercial kits .At the end of 10th week of the experiment period, blood samples were collected morning before adding feed, from the margin ear of bucks after disinfecting with a sanitizer solution in sterilized tube. Then the blood samples were centrifuged at 700The cortisol (MBS2005085) and testosterone (MBS704954) enzyme-linked immunosorbent assay (ELISA) MyBioSource kits  were employed in quantifying the concentrations of cortisol and testosterone, respectively, following the manufacturer’s protocols.p < 0.05, according to the following statistical model: One-way model: YiK = μ + Xi + eik where: YiK = the response variable; μ = the overall mean; Xi = the fixed effect of treatment ; eik = the residual error. Figures were fitted by the SigmaPlot .The experimental data were subjected to a one-way analysis of variance analyzed (ANOVA) via the GLM procedure using Statistical Analysis Software . Each buck rabbit is an experimental unit that provides an independent measurement. All percentage data were arc-sine transformed prior to the approximate normal distribution. The Tukey test was used to compare the differences between the means when significant F values were observed at the levels of All experimental procedures and handling of buck rabbits were approved by the Alexandria University Institutional Animal Care and Use Committee.Figure P < 0.05) was increased by inclusion zinc with different forms in heat stressed bucks’ rations compared to the control group and this effect was significant with zinc Met and ZnO NPs only.The data in Table The antioxidant status of seminal plasma of heat stressed bucks such of SOD and CAT levels Fig.  were sigAs described in Table 39. The weekly THI values clearly indicated that the buck rabbits suffered severe thermal stress. Zinc is an effective antioxidant and plays a vital physiological role for animal health, synthesis of more than 300 enzymes involved in metabolism process40, synthesis proteins, immune system activation, bone development41, DNA synthesis, cell division, gene expression, and biological functions42. In addition to reducing the negative impact of both cold and heat stressors43. Zinc methionine is organic forms of Zn are generally bound to methionine amino-acid complex, it may be better absorption and more available to animal by interaction with other elements, especially Cu, Fe, and Ca44. Nano-zinc oxide is smaller than 100 nm causes increasing absorption rate and availability40, and is more biologically effective45 by bypassing conventional physiological ways of nutrient distribution, and transport across tissue and cell membranes, as well as protecting compounds against destruction before reaching their targets19, and reaching target cells more quickly than ordinary minerals with larger particle size20. At the same time, using nano-Zn oxide is useful to avoid excessive supplementation with Zn in animal diets and lowest the accumulation of Zn in soils after intensive animal farming46, thus reducing the potential risks for environmental pollution47.Temperature humidity index (THI) is used to evaluate the degree of heat stress by the combined both ambient temperature and relative humidity estimate the severity of heat stress. This index is widely used in hot and humid areas around the world to assess the impact of heat stress48 and accordingly measure the degree of discomfort/comfort or adaptability of bucks. Our result revealed that all sources Zn supplementation significantly decrease RR and RT values of heat stressed bucks on 1st month and RR only on 2nd month. The positive impact of zinc on physiological parameter of heat stressed buck may be due to that zinc reduced stress on the rabbits by enhancing the bioavailability of other minerals49. The insignificant RT values in the 2nd month of all zinc treatments may be due to the development of metabolic mechanisms of all experimental bucks to adapt to heat stress to a tolerable level.Physiological parameters such as respiration rate and rectum temperature are considered direct indexes of climatic stressThe initial and final body weights of the heat-stressed bucks did not differ in all experimental groups. Although the body weight change of all zinc treatments was significantly greater compared to the control bucks, that cleared that all zinc treatments alleviated the negative effect of heat stress.51 who concluded a significant increase in semen volume, total live sperm concentration and sperm mobility due to zinc sulphate dietary supplementation in heat-stressed rabbit males. Increased semen volume of Zn-s treatment may contribute to increased prostatic fluid9. Furthermore, nanozinc significantly increased each of sperm concentration, sperm viability, and initial fructose compared to the control group could be due to improvement in buck antioxidant status52. Similar results were found by45 who reported that nanoparticles have biological effects that improve male fertility by enhancing sperm quality. Also,53 observed that supplemented young rams’ diet with 50 mg/kg or 100 mg/kg nano zinc practical improved epididymal semen quality. Increasing the percentage of sperm viability of nanozinc bucks may be due to improvement the functionality of sperm plasma membranes without any significant effect on motility parameters54. Furthermore, the improvement in sperm concentration of bucks fed a diet supplemented with nano-zinc could be due to increased antioxidant enzymes activity of testicular tissue55 or seminal plasma in the current study that protects sperm against oxidative stress. In addition, Zn-Met significantly enhanced the viability sperm percentage and initial fructose in the present findings.Zinc sulphate treatment significantly improved semen volume, sperm concentration and sperm viability; a similar improvement was obtained by56. Other semen characteristics such as pH and motility  were not differ among experimental groups. Our results agree with those of57 and58 who reported that supplemental organic and non-organic zinc improved quantitative and qualitative characteristics of semen reproductive efficiency59 and fertility by lower blood kidney parameters that decrease ROS production and increased progressive motility, acrosome reaction and capacitation60. Viable sperm percentage did not effect by all zinc treatments in the current results, this observation has a harmoniousness with previous studies of61. In general, zinc affects reproductive characteristics by stimulating and protecting the germinal epithelium of seminiferous tubules and promoting the production and secretion of testosterone62, as shown in the present findings, and therefore influences spermatogenesis63. Besides, the positive result of all zinc treatments on sperm concentration and spermatogenesis may be due to the production of sperm necessitates extensive cell division by influencing mitotic and meiotic cell divisions, synthesis of DNA and RNA by improving the activity of DNA and RNA polymerase (Zn-containing enzymes)64. Increasing initial fructose in all zinc treated bucks as compared to the control bucks may be attributed to the effect of treatments on fructogenesis in seminal vesicles of heat stressed rabbits.Additionally, the membrane of rabbit sperm cells is rich in polyunsaturated fatty acids that make sperm susceptible to lipid peroxidation65 who reported that Zn supplementation had a beneficial effect on the anti-oxidative status of seminal plasma, which may provide better protection to spermatozoa from oxidative damage, and also improve the concentration of testosterone as is shown in the current study which may play a role in the production of good quality semen.Antioxidant status such as SOD and CAT levels of the seminal plasma of heat-stressed bucks in the present findings were significantly improved by different sources of zinc treatments. Although zinc methionine supplementation was more effective in reducing the seminal plasma TBAR concentration compared to other groups. These results agreed with those presented by66. Our results were in agreement with68 who found that supplementation with ZnO-NP in rabbit diets significantly reduced serum levels of AST and ALT activities compared to the control group. All zinc treatments improved kidney function as reflected in the decrease in creatinine values. The current results were agreed with70 that found the diets supplemented with 50 and 100 mg/kg of Zn-Met significantly decreased blood creatinine concentration compared to the control group. On the other hand, the result disagreed with71 who reported that plasma creatinine concentration did not change in rabbit fed diet supplemented with Zn.Liver functions were significantly improved as AST and ALT concentrations were reduced in Zn-Met group compared to the control group with no significant effect of other zinc treatments. The positive effect of Zn treatments on semen quality may be due to the revealed negative relationship between liver enzymes such as ALT and GGT and sperm concentration, total testosterone and LH due to the formation of pro-oxidant species formation72. Dietary supplementation of different Zn sources in the current study improved the serum antioxidant status (SOD and TAC levels) with no significant effect between ZnO NPs and control groups. The improvement in blood antioxidant status may be due to zinc being considered an effective antioxidant by having catalytic, coactive, or structural functions in different enzymes such as SOD, which controls many physiological processes such as metabolism and immune function73, or by inhibiting the oxidation of macromolecules such as DNA and proteins17 or by maintaining the activities of radical scavenging enzymes40. The improvement in blood antioxidant status may be associated to the enhancing in seminal antioxidant status as mentioned earlier in our study, which illustrated that zinc prevents the oxidative damage to the sperm cells and thereby improve the fertility11. From Fig. 74 who showed that plasma TBARs and plasma lipid peroxides concentration was reduced significantly by zinc supplementation in rabbit diets.Heat stress decreases antioxidant status, which is one of the most important physiological changes in response to high ambient temperature in animals that increases oxidative stress and immune suppression75. However, zinc had an effect on the severe heat stress by decreasing cortisol levels which observed in the current study, whereas all zinc treatments declined serum cortisol levels. Testosterone as a sex hormone has a positive impact on semen quality and reproductive physiology76. In the present findings, all zinc forms led to elevated serum testosterone concentration of heat stressed bucks, which reflected the improvement in semen characteristics which was in consistency with previous studies. Many studies supported our results on improving blood testosterone concentration61, which helps in building protein, and is necessary for sexual desire and erections77.Animals that exposed to ecological stressors such as extremes of temperatures, neurons in the CNS are stimulated, that activated of the hypothalamic pituitary adrenal axis leading to stimulate the cortex of the adrenal gland to produce and release corticosteroids (cortisol)st month of experiment, and seminal plasma antioxidant (SOD and CAT) of heat stressed bucks with the three forms of dietary Zn supplementation. Furthermore, semen quality in terms of sperm concentration, viability, and initial fructose improved significantly with ZnO-NPs supplementation. Zinc methionine supplementation significantly improved liver function and reduced seminal plasma TBARs. Both ZnO-NPs and Zn-Met improved seminal TAC and blood testosterone with reduced blood cortisol levels. Therefore, it concluded that the recommended forms of Zn in the dose of 30 mg/kg diet are zinc methionine and ZnO-NPs, rather than ZnS during the severe heat stress conditions to improve semen quality, and serum and seminal antioxidant status of buck rabbits.Current data revealed a significant positive improvement in body weight change, blood kidney function (creatinine), serum antioxidant (SOD and CAT), physiological parameters, especially in the 1


This study aimed to predict the fracture resistance of a mandibular first molar (MFM) with diverse endodontic cavities using finite element analysis (FEA).Five experimental finite element models representing a natural tooth (NT) and 4 endodontically treated MFMs were generated. Treated MFM models were with a traditional endodontic cavity (TEC) and minimally invasive endodontic (MIE) cavities, including guided endodontic cavity (GEC), contracted endodontic cavity (CEC) and truss endodontic cavity (TREC). Three loads were applied, simulating a maximum bite force of 600 N (N) vertically and a normal masticatory force of 225 N vertically and laterally. The distributions of von Mises (VM) stress and maximum VM stress were calculated.The maximum VM stresses of the NT model were the lowest under normal masticatory forces. In endodontically treated models, the distribution of VM stress in GEC model was the most similar to NT model. The maximum VM stresses of the GEC and CEC models under different forces were lower than those of TREC and TEC models. Under vertical loads, the maximum VM stresses of the TREC model were the highest, while under the lateral load, the maximum VM stress of the TEC model was the highest.The stress distribution of tooth with GEC was most like NT. Compared with TECs, GECs and CECs may better maintain fracture resistance, TRECs, however, may have a limited effect on maintenance of the tooth resistance. The primary goal of endodontic treatment is the long-term retention of functional teeth but increasing the long-term retention rate of endodontically treated teeth (ETT) is still a great challenge. For decades, dental practitioners have been looking for ways to enhance fracture resistance, because tooth fracture is considered one of the main reasons resulting in the extraction of ETT . BenefitEven 10 years after the first proposed application and the religious support from proponents and influencers in the field of endodontology, MIE is still controversial because many critical aspects still remain to be studied, and no clear evidence shows that MIE is better than traditional endodontics in fracture resistance –5. Some Finite element analysis (FEA) is a promising theoretical stress analysis method because the sample demand is small and the stress inside the model can be displayed, indicating the fracture risk areas intuitively . AdditioTo study whether or which MIE cavity can better maintain the resistance of tooth fracture after endodontic treatment, different MIE cavity models of a mandibular first molar (MFM) were established using a three-dimensional finite element method. A 3-point vertical/lateral static force load simulating normal masticatory force and an 8-point vertical static force load simulating maximum bite force were applied on the occlusal surface of different models. The null hypothesis is that different endodontic cavities behavior similar on stress distribution for an MFM. The maximum von Mises (VM) stress and three cross sections of different models were evaluated.With consent of the patient and approval by the ethics committee of the School of Stomatology of the fourth Military Medical University , a fresh, intact, non-carious, lightly wear, mature human MFM with three canals was obtained and scanned with a micro–computed tomographic scanner . The scanning parameters were as follows: 80 kV, 10 W, and 1.5 μm slice thickness. The image data were exported in digital imaging and communications in medical (DICOM) format. A MIMICS 16.0 interactive medical imaging system  was used to identify the different hard tissues and design different endodontic cavities. Three-dimensional (3D) objects (enamel and dentin) were automatically created in the form of masks and exported as STL files. These files were refined with reverse engineering software . The enamel and dentin were combined using 3-Matic Research 12.0 .In this research, a natural tooth (NT) and 4 endodontically treated MFM models were investigated.According to the volumes of removed coronal dentin and PCD, 4 cavity preparations were adopted, including the GEC , CEC 2]2], TREC The pathways of the GEC model were three separating cylinders in the direction of the coronal 1/3 of every root canal so that the PCD could be preserved as much as possible. The CEC outline was determined with three cylinders straight into the root canal orifices meeting the occlusal surface. The outline of TREC was determined with three cylinders straight to the root canals and vertical to the partially overlapping occlusal surface.With the appropriate modifications of the microcomputed tomographic data, 3-dimensional models of endodontically treated teeth were created based on the cavities described previously. Root canal therapy was simulated by computer according to a standardized procedure . The disThe periodontal ligament was generated by creating a uniform 0.25 mm layer around the root . MeanwhiThe FEM was performed using HyperMesh 14.0  to calculate VM stress in the enamel and dentin. The analysis was based on the following assumptions:Each material was presumed to be homogeneous, isotropic and linearly elastic.There was perfect bonding between each component.There was no flaw in the initial model.There were rigid constraints on the base and lateral surfaces of the alveolar bone.The force loading processes  were as Load A : The models received a vertical static force load of 225 N (N) in total to simulate a normal vertical mastication force load. The force load was applied to the occlusal surface at 3 registered contact points  : A static force load of 225 N was applied laterally (45° to the tooth axis) at 3 registered contact points  to simulate the lateral mastication force load : A static force load of 600 N was applied vertically to 8 contact points registered  on the occlusal surface to simulate the maximum bite force load , the pulp chamber floor (PCF), and the apical foramen (AF) was investigated . A conveAll five three-dimensional finite element models were established successfully. The VM stress diagrams of all the models are listed in Fig. 3, 23.17 mm3, 28.34 mm3 and 45.57 mm3.By calculating the difference in volume cavity preparation between ETT models and NT, the volumes of removed dentin in GEC, CEC, TREC, and TEC were 33.16 mmUnder the vertical 3-point force load, the maximum VM stresses in the NT, GEC, CEC, TREC and TEC models were 203.1 MPa, 208.5 MPa, 210.2 MPa, 229.6 MPa and 210.6 MPa, respectively  using extracted natural teeth, FEA allows for better control of variables and excludes bias due the experimental manipulation such as practitioner experience and variations between teeth. Therefore, the use of FEA rather than ESA has been advocated in the methodological design of studies on the effect of MIE cavity on the fracture resistance of ETT. However, Within the limitations of this study, the cervical region was a stress concentration area in all models. The stress distribution of tooth with GEC was most like NT. Compared with TECs, GECs and CECs may better maintain fracture resistance, TRECs, however, may have a limited effect on maintenance of the tooth resistance. GECs may be superior to CECs for MFMs in that GECs may lead to more restorable fractures.


Introduction: Mechanic power output (MPO) and oxygen consumption (VO2) reflect endurance capacity and are often stated relative to body mass (BM) but less often per skeletal muscle mass (SMM). Rating of perceived exertion (RPE) has previously shown conflicting results between sexes at submaximal intensities. Individual body composition, however, largely differs due to sex and training status. It was the aim of this study to evaluate RPE of untrained and trained individuals of both sexes considering body composition and to estimate whether RPE could be improved as a tool to determine endurance capacity.Methods: The study included 34 untrained adults  and 29 endurance trained  who were measured for body composition  and tested on a treadmill  for aerobic capacity  in an all-out exercise test applying the Bruce-protocol. VO2, MPO, heart rate (HR), and RPE were obtained at each exercise stage. VO2 and MPO were calculated per BM and SMM. RPE values were correlated with absolute VO2 and MPO, as well as relative to BM, and SMM. HR values and the parameters’ standardized values served for comparison to standard procedures.Results: VO2 and MPO were higher in men compared to women and in trained compared to untrained participants. No differences between groups and sexes exist when VO2 and MPO were calculated per BM. When calculated per SMM, VO2 and MPO indicate opposite results already at low intensity stages of exercise test. RPE values had highest correlation with MPO per SMM (R2 = 0.8345) compared to absolute MPO (R2 = 0.7609), or MPO per BM (R2 = 0.8176). Agreement between RPE and MPO per SMM was greater than between RPE and HR (p = 0.008).Conclusion: Although RPE represents a subjective value at first glance, it was shown that RPE constitutes a valuable tool to estimate endurance capacity, which can be further enhanced if individual body composition is considered. Furthermore, MPO and VO2 should be considered relative to SMM. These findings might help to avoid over-exertion, especially among untrained people, by adjusting the training intensity for each subject according to the individual strain evaluated in an exercise test based on individual body composition. Both pamulation . Especiamulation  in ordermulation . In contectively .2 based on individual body composition at different exercise intensities. It was the aim of this study to fill this gap to gain better insight on how healthy adults of different sex and training status perceive physical strain. Moreover, the study aimed at evaluating whether VO2 and MPO based on individual SMM will provide additional knowledge in determining RPE. Such analysis may offer an individualized recommendation of training intensities based on individual body composition. To address these aims, three hypotheses were constructed: 1) Objectively measurable parameters of physical strain will differ between the sexes and training status dependent on their representation either as absolute values, or relative to body mass or SMM; 2) Individually reported RPE show a greater correlation to VO2 and MPO when they are calculated per SMM; 3) Compared to the agreement between RPE and HR, the agreement between RPE and VO2 and MPO relative to SMM will add additional knowledge.So far, no attempt has been made to evaluate the influence of sex and training status on individual RPE and its relation to objectively measurable physical strain parameters such as MPO and VO2), endurance training status (either being endurance trained or not) and being of general and cardiac health (including an asymptomatic electrocardiogram at rest). Based on their biological sex  and based on self-reported weekly training hours, as described below, participants were divided in four groups: women untrained , women trained , men untrained , and men trained . Classified as trained (T) were participants who exercised at least three times per week for a minimum of 1 h per session for at least 1 year prior to the study, whereas subjects who did none or just occasional exercise were classified as untrained (UT). The recruitment process is indicated in A number of n = 63 adults were recruited as part of a larger investigation through online and in-print advertisements briefly outlining the study as described in previous reports . InclusiThe potential study participants had several weeks to pose questions and to decide to partake in the study. The study was approved by the Charité Ethics Board (IRB-number: EA1/154/18). All conducted measurements and procedures complied with the Declaration of Helsinki  concerning the treatment of human subjects. All participants gave their informed written consent.All tests were performed from 9 a.m. to 12 p.m. under standardized conditions  between February and April 2019. Participants gave information about their training and received a description of the test procedures. Blood pressure and electrocardiogram (ECG) were measured from each subject and evaluated by a medical doctor along with a short medical anamnesis to exclude contraindications for a maximal exercise test. Resting heart rate (RHR) were obtained through a mobile HR monitor  that has been validated for scientific use , while p2max defined as the maximum amount of oxygen to be utilized , which has been validated for scientific use (2) × Velocity (m/s) × Treadmill Grade (%). The workflow is visualized in Subsequently, an exercise test was carried out on a treadmill  that has been validated for scientific application , using tutilized . Continuific use . Velocitific use . Again, ific use . In addiific use  and reacific use . In the ific use . MPO for2max of healthy young adults  and regarding data on exercise test performance: maximal performance time (min), VO2max (ml/kg/min), maximal MPO (Watt), maximal HR (bpm), and maximal RPE value. Effect sizes are stated as “Hedges’ g” values for differences between groups of unequal sample size  and RHR, normal distribution was tested with Shapiro-Wilk test  and variple size . For nonple size .2, mean achieved MPO values, and mean RPE values were determined at each stage of the applied Bruce protocol. Mean achieved VO2 and MPO values were then calculated for each participant per BM and per SMM by dividing the respective VO2 and MPO values by the obtained values of BM and SMM of each participant. At each stage of the Bruce protocol (from rest to stage 7), these values were tested for differences across all four groups using Analysis of Variance (ANOVA) (post hoc Multiple Comparison Procedures (Holm-Šídák corrected tests) (post hoc Multiple Comparison Procedures (Dunn’s corrected tests) were applied   and postd tests)  to compad tests)  and non- applied .2, VO2 per BM (VO2BM), VO2 per SMM (VO2SMM) and MPO, MPO per BM (MPOBM), MPO per SMM (MPOSMM), and HR, being the independent variables, respectively.Simple linear correlation analyses (Pearson)  were per2, VO2BM, VO2SMM and MPO, MPOBM, MPOSMM, and HR, obtained at each exercise stage, were standardized to calculate their “z-scores” by subtracting the mean of each parameter from all its raw values and subsequent division of the subtraction results by the standard deviation of each parameter, so that their mean was zero and their standard deviation was one  and method two  were then statistically compared with the absolute values of differences between RPE and HR through a post hoc Multiple Comparison Procedure (Holm-Šídák corrected tests).All values of RPE, VO was one . This st was one , where tBM, RPE and MPOSMM, and RPE and HR, to graphically represent their agreement. The statistical analyses and graphic representations were performed using “GraphPad Prism” for Windows, version 9.3.1 ; sample size and statistical power were calculated using “Systat SigmaPlot” for Windows, version 14.5 . Data are reported as mean values ± standard deviation (SD) and statistical significance was assumed at a p-value of <0.05.For graphic representation, boxplots of MPO  and of RPE values were plotted to indicate group effects between the four groups  at each Bruce level. In addition, Bland-Altman plots were added for standardized values of RPE and MPO, RPE, and MPOversus trained groups of both sexes, there were no differences for age, BM, height, and BMI. A difference was observed for RHR, with significantly lower values (>10 bpm) among the trained individuals, which corroborated the interview results taken to allocate the participants to either UT or T groups. Furthermore, percentages of FM and SMM were significantly different with lower values for FM and higher values for SMM for the trained groups.The anthropometric and RHR data are presented in 2max, and greater MPO than women. There were no differences between sexes for maximal attained RPE and maximal HR values among neither of the trained or untrained group, except for UT where men showed greater maximal HR than women. The trained group among both sexes achieved significantly greater maximal performance time, greater MPO, and higher values of VO2max than the untrained group. There were no significant differences between UT and T for maximal attained RPE and maximal HR values among neither sex. Exercise performance results are presented in All participants reached at least 95% of the predicted maximal heart rate, while a mean maximal respiratory quotient of 1.11 (±0.08) was reached. At absolute values, men achieved significantly greater maximal performance time, higher values of VO2 indicate significantly greater values for men compared to women and for trained individuals compared to the untrained. Notably, calculated per BM, VO2 does not show significant differences between the groups except for stage 5. Calculated per SMM, the results of VO2 indicate significantly greater values for women compared to men and for untrained compared to the trained individuals, at test stages 1–4. 2  for each group at each stage of the applied Bruce protocol and the results of the performed ANOVA for differences across all groups, while The results of absolute VOBM does not show significant differences between the groups, at none of the exercise stages.The results of absolute MPO indicate significantly greater values for men compared to women and for trained individuals compared to the untrained, at each stage of the exercise test. MPOSMM indicate significantly greater values for women compared to men and for untrained compared to the trained individuals, at test stages 1–6. Results of MPOThe results of RPE indicate significantly greater values for the untrained compared to trained individuals, at the stages 1, 2, and 4 to 6. p-values indicate a highly significant correlation between the RPE and the independent parameters. With regards to VO2 being the independent variable, the correlation coefficients (R) and coefficients of determination (R2) indicate a greater determination of the dependent variable “RPE” in dependence of “VO2BM” (R2 = 0.8103) when comparing it with the dependence of “VO2 absolute” (R2 = 0.7250) and a slightly greater value in dependence of “VO2SMM” (R2 = 0.8171). With regards to MPO being the independent variable, the coefficients of determination (R2) indicate a greater determination of the dependent variable “RPE” in dependence of “MPOBM” (R2 = 0.8176) when comparing it with the dependence of “MPO absolute” (R2 = 0.7609) and an even greater value in dependence of “MPOSMM” (R2 = 0.8345). The correlation between RPE and HR yielded an R2 of 0.7745. The results of the correlation analyses are shown in All 2, and RPE, and MPO, respectively, are smallest when they are calculated per SMM. The standard deviations of differences between RPE and MPOBM (0.4324) and MPOSMM (0.4179) are significantly smaller than the standard deviation of differences between of RPE and HR (0.4905), which is graphically represented in The results of the Bland-Altman analyses of the standardized values are shown in 2 based on individual body composition at different exercise intensities to gain better insight on how healthy adults of different sex and training status perceive physical strain.It was the aim of the study to evaluate the influence of sex and training status on individual RPE and its relation to objectively measurable physical strain parameters such as MPO and VO2 and MPO are higher in men compared to women and in trained compared to untrained participants, but when calculated per SMM, VO2 and MPO indicate opposite results already at low intensity stages; 2) RPE can be closely related to objectively measurable parameters of physical strain, like the MPO and VO2, and—as a key finding of this study—to a considerably greater degree when these parameters are calculated per SMM where RPE values had highest correlation with MPO per SMM compared to absolute MPO, or MPO per BM; and 3) within the 15-grade Borg scale, the agreement of RPE with MPO relative to SMM was higher than the agreement of RPE and HR.The three formulated hypotheses of this study were confirmed by the results 1) VOWithin each group, participants were of comparable age and RHR. There were no differences between the untrained and trained subjects regarding age, BM, height, and BMI, which can thusly be construed as comparable homogenous groups regarding these parameters. Among the trained, there were lower FM, higher SMM, and lower RHR, thus reflecting their better endurance training status .2max, and maximal MPO, which was to be expected from previous literature on test performance  as subjlic rate . The lat2 and MPO. Interestingly, the correlation coefficient and coefficient of determination (R2) are highest, when the correlation is calculated for VO2SMM and MPOSMM, indicating muscle mass as the most relevant factor: while only 76.1% of the variation of the RPE values is explained by absolute MPO, 83.4% of the variation of obtained RPE values is explained by MPOSMM, while merely 77.5% of the variation of the RPE values is explained by HR, which is interesting since HR has been the basis for the RPE scale  but as a mathematical tool to test the agreement of the obtained values in order to leverage and enhance the performed correlation analyses, since all correlation results were highly significant. After standardization of the obtained values and subsequent application of the Bland-Altman method, greatest agreement was shown between RPE and VO2SMM and RPE and MPOSMM, respectively. Surprisingly, the agreements between RPE and MPOBM, and RPE and MPOSMM, respectively, were significantly greater than the one of RPE and HR, on which the RPE 15-grade scale was originally based upon  obtain a prior analysis of their body composition to be able to calculate MPOSMM for generating individualized exercise guidelines to avoid over-exertion. On the other hand, the results underscore the validity of the RPE scale, and that this individual and subjective “inner voice” shows high correlations and agreements with other objective strain parameters, which warrants the use of the RPE during exercise testing, prescription, and performance.To summarize, the results regarding VO2, and MPO.All study participants were young and healthy adults, therefore the use on other populations of different age, or different health conditions might not be directly applicable, especially in patients encumbered by cardio-pulmonary limitations or obesity. Further studies seem warranted in other exercise settings as well as among populations other than young healthy adults. Further efforts should include body composition evaluations based on other means than bioimpedance analyses, which might be not readily available in each instance. In addition, future studies might be directed to investigate allometric exponents for concurrent size descriptors , such as proposed in a previous work , along w


Background: Studies addressing the utility of hemoglobin A1c (HbA1c) levels in predicting surgical complications have reported mixed results. In practice, many surgeons use HbA1c cutoffs to determine a patient’s eligibility for elective surgery. The literature is especially limited in evaluating HbA1c as a risk factor in elective hand and upper extremity surgery. This study aims to evaluate the association of elevated HbA1c levels with the risk of postoperative complications in elective hand and upper extremity surgeries.Methods: We performed a chart review of patients who underwent these elective operations performed by a single surgeon at a single institution. The outcomes of 930 surgeries were collected up to three months postoperatively, 334 of which had pre or postoperative HbA1c levels recorded. All 930 surgeries were evaluated for association between diabetes mellitus (DM) and complication rates using Fisher’s exact test, absolute risk, odds ratio, and regression analysis.Results: DM alone was not associated with an increased risk of postoperative complications. In diabetic patients with both diagnosed DM and HbA1c > 10%, the absolute risk of complications was significantly higher. None of the other HbA1c categories (between 6.5% and 10%) were associated with significantly higher odds of complications in patients with diagnosed DM.Conclusions: In the context of elective hand and upper extremity surgery, glycemic control (measured by HbA1c) should be included as an element of the surgical preparedness algorithm, rather than an independent disqualifying factor. Historically, diabetic patients are known to have an increased risk of postoperative complications compared to the non-diabetic population. These complications include but are not limited to infections, impaired wound healing, cardiovascular events, venous thromboembolism, and mortality. Given that poor glycemic control has been identified as a significant risk factor, hemoglobin A1c (HbA1c) has been used as a biomarker to evaluate patients’ preparedness for surgery [The American Diabetes Association recommends a target of HbA1c < 7% for diabetic patients , and somOne of the most common and concerning surgical complications, especially in patients with poor glycemic control, is infection. In 2018, Sharma et al. performed a prospective cohort study following diabetic and nondiabetic surgical hand and forearm infections over three years. In diabetic patients, the need for recurrent drainage was not significantly different between those with good versus poor baseline glycemic control (HbA1c > 9.0%). However, patients with poor inpatient glycemic control (average inpatient blood glucose of >180 mg/dL) had a significantly higher need for additional drainage [The correlation of HbA1c levels with postoperative complications has been documented in a variety of surgical procedures with mixed results. In a systematic review of non-diabetic patients, Karimian et al. analyzed the results of six observational studies. Four studies reported a significant correlation between preoperative HbA1c levels and postoperative complications. Only two of these four reported increased infection rates with elevated HbA1c, and two reported no significant difference . None ofIn hand surgery, HbA1c data have been examined to determine the association between increased HbA1c levels and the development of trigger digit after carpal tunnel release (CTR). In 2014, Grandizio et al. evaluated 1,217 cases of CTR, 214 of which were performed on diabetic patients. Of the non-diabetic cases, 3% developed trigger digit within six months of CTR, and 4% within one year. Of diabetic patients, 8% developed trigger digit within six months and 10% within one year. Thus, diabetes was identified as a significant risk factor for developing trigger digit after CTR. Interestingly, no significant association was found between HbA1c level at the time of CTR and the likelihood of developing TD [Elective surgeries of the hand and upper extremities such as carpal and cubital tunnel release, arthroplasty, and tendon transfer have been shown to significantly improve function, pain, and quality of life for patients -14. PhysApproval for this study was obtained from our Institutional Review Board . Charts were collected for all patients who underwent common elective hand and/or upper extremity surgeries performed by a single fellowship-trained hand surgeon from 2012 to 2018 at a single hospital Tables , 4. PatiFollow-up clinic notes, diagnoses, and medications were evaluated three months postoperatively for the incidence of postoperative complications. Complications in our cohort were defined as infection, dehiscence, and unimproved or worsening of preoperative symptoms. Comorbidities, including diagnosed DM, peripheral neuropathy (PN), and macrovascular disease , were recorded.HbA1c values were analyzed using cutoffs  and categories . The absolute risk, odds ratios, and Fisher’s exact tests were calculated to evaluate the risk of postoperative complications in each group Tables , 4. PresRegression analysis was also performed to assess the degree of correlation between the incidence of elevated HbA1c, DM, duration of DM , and macrovascular disease with postoperative complications Table . PearsonA total of 930 patients were included in our study. A total of 502 patients underwent more than one procedure Table  in a sinThere were 22 complications at three months postoperatively in our entire cohort n = 930). Infection accounted for 31.8% (7/22) of complications, and the remaining 68.2% (15/22) experienced no improvement or worsening of preoperative symptoms. A list of procedures paired with their respective postoperative complications can be found in Table 0. InfectIn the preoperative HbA1c group, none of the HbA1c cutoffs were significantly associated with an increased risk of complication. Diagnosis of DM or PN also failed to increase risk Tables , 5. For Regression analysis did not show a significant correlation between any HbA1c cutoff, DM, DM duration, or macrovascular disease with the incidence of postoperative complications Table .Principle among the concerns of operating on patients with high HbA1c levels is the risk of postoperative infection. HbA1c is a measure of glycemic control, which is frequently a concern for patients with DM. The hyperglycemic environment in diabetic patients can cause immune dysfunction by suppressing mechanisms needed to fight infection, including T-lymphocyte response, neutrophil function, and secretion of inflammatory cytokines . These mA study by Endara et al. measured HbA1c and blood glucose levels five days before and after high-risk surgical closure. Univariate and multivariate analyses were performed to determine the correlation of elevated HbA1c levels (above 6.5% and 7.0%) with incidence of dehiscence, infection, and reoperation. Results showed that while dehiscence increased as HbA1c increased, there was no significant correlation between either cutoff and increased rates of infection and/or reoperation .Given the variability in protocols and reported outcomes regarding HbA1c, recent studies have investigated additional comorbid conditions as potential risk factors. One prospective study evaluated 2,060 foot and ankle surgeries over a period of four years to determine the correlation of DM and PN with postoperative infection . InvestiIn a separate study evaluating the relationship of surgical morbidity to glycemic control in elective foot and ankle surgery, Domek et al. reviewed 21,854 diabetic patients with HbA1c measurements < one year prior to elective foot and ankle surgery . The posHbA1c is not the only metric that has been questioned as a determinant for surgical candidacy. Hard cutoffs are often used for weight, BMI, and cigarette smoking as well . GlycemiEven when resources are available, recommended levels of biomarkers such as HbA1c are notoriously difficult to achieve. In a study assessing the effects of literacy, numeracy, and medication adherence on glycemic control, Huang et al. found that medication adherence was only weakly correlated (rho correlation coefficient: 0.238) with glycemic control (HbA1c ≤ 7.0%) .Setting hard cutoffs for HbA1c and other comorbid domains serves to deindividualize patients, and could lead to discriminatory practice against patients who would have tolerated the procedure well. It is well known that lower socioeconomic status (SES), as well as minority race and ethnicity, can negatively influence clinical outcomes such as glycemic control in diabetic patients . InteresGiven the nature of blue-collar jobs, which often include manual labor, it is possible that low SES patients may benefit more from these operative interventions, as physical aptitude directly affects their livelihood. By allowing a single risk factor without consensus support in the literature to bar them from treatment, surgeons may unknowingly discriminate against these populations.The present study is limited by the variable timing of HbA1c testing. Ideally, HbA1c measurements are taken < three months prior to surgery. HbA1c levels at ≤ six months and ≤ one year are indirect measures of preoperative glycemic control and are not intended to serve as a surrogate for HbA1c obtained within the three-month window. The majority of procedures included in our study have relatively low risk . Future studies may better address procedures such as arthroplasties and tendon grafting - where infection and wound dehiscence are more likely to occur. Additionally, a small sample of patients in the higher HbA1c cutoff groups is prone to beta error and may be insufficient to draw conclusions regarding these patients in our cohort. Given the retrospective nature of this study, variables such as the duration of DM may not be precise. The date that DM is detected and documented in the electronic medical record (EMR) does not necessarily indicate the actual onset of DM.The results of our study add to a growing body of literature addressing the utility of HbA1c as a preoperative qualification for surgery. The data we present indicate that, throughout a wide range of cutoff points, elevated HbA1c is not an independent predisposing factor for postoperative complications in elective procedures of the hand and upper extremities. Given our findings and those of similar studies in the literature, we recommend that surgeons regard HbA1c as one piece of the surgical candidacy algorithm rather than an independent gatekeeper for elective procedures.


Caesarean scar pregnancy (CSP) is a special type of ectopic pregnancy with a high risk of massive haemorrhage. Few studies have focused on the efficacy of prophylactic abdominal aortic balloon occlusion as a minimally invasive method in caesarean section. This study aimed to evaluate the effectiveness and safety of prophylactic abdominal aortic balloon occlusion for patients with type III CSP.This was a prospective cohort study. Patients with type III CSP in the First Affiliated Hospital of Zhengzhou University from January 2020 to June 2022 were enrolled. Eligible patients received prophylactic abdominal aortic balloon occlusion (defined as the AABO group) or uterine artery embolization (defined as the UAE group) before laparoscopic surgery. Clinical outcomes included intraoperative blood loss, body surface radiation dose, hospitalization expenses, and time to serum β-hCG normalization, and safety were also assessed.P = 0.264). The body surface radiation dose of the AABO group was much lower than that of the UAE group . The AABO group also had lower hospitalization expenses than the UAE group . The average time to serum β-hCG normalization in the AABO group was 28.9 ± 3.21 d, which was similar to that in the UAE group . In addition, the incidence of adverse events in the AABO group was lower than that in the UAE group .A total of 68 patients met the criteria for the study, of whom 34 patients were in the AABO group and 34 patients were in the UAE group. The median intraoperative blood loss in the AABO and UAE groups was 17.5  and 10  mL, respectively (Prophylactic AABO was equally as effective as UAE in patients with type III CSP but was safer than UAE during and after the operation. Caesarean scar pregnancy (CSP) is characterized by the presence of a gestational sac embedded in the scar from a prior caesarean delivery . The preTermination of CSP is required. A variety of therapeutic strategies have been described for managing CSP, including drugs, uterine artery embolization (UAE), curettage, and laparoscopic or transvaginal surgery . HaemostIncreasing evidence suggests that UAE can be minimally invasive in CSP in that it can be performed with laparoscopy, hysteroscopy, or local resection , 9. UAE This study aimed to evaluate the effectiveness and safety of prophylactic AABO and UAE for patients with type III CSP.This was a prospective cohort study. Patients with CSP were enrolled from January 2020 to June 2022 at the First Affiliated Hospital of Zhengzhou University. The inclusion criteria were as follows: (1) aged 18 years or older; (2) 3–16 weeks of gestation; (3) diagnosis and confirmation of type III CSP  by both transvaginal colour Doppler sonography (TVCDS) and MRI. The exclusion criteria were as follows: (1) presence of severe vaginal bleeding (emergency); (2) previous history of uterine surgery other than caesarean section; (3) unsuitable conditions making the patient ineligible for hysteroscopic surgery, including bleeding disorder and impaired renal or hepatic function; and (4) refusal to participate in the study. Eligible patients were assigned to the AABO group or the UAE group based on the preconditioning regimen they received.This study was approved by the institutional review board of the ethics committee of the First Affiliated Hospital of Zhengzhou University (2021-KY-0209-004). Written informed consent forms were obtained from each enrolled patient.All patients underwent standardized procedures to resolve the caesarean section scar pregnancy. All patients underwent laparoscopic removal of gestational tissues and repair of uterine caesarean scar defects after prophylactic UAE or AABO. Balloon tamponade and UAE were performed by the same radiologist and the same assistants. All operations were performed by the same surgeon and the same surgical assistants and nurses using endoscopic instruments.Patients with type III CSP in the AABO group were treated with abdominal aortic balloon occlusion prepositioning under digital subtraction angiography (DSA) while undergoing hysteroscopy and negative pressure suction. Under local anaesthesia, femoral artery puncture was performed, an 8 F sheath tube was inserted, and abdominal aortography was performed through the sheath tube. The morphology of the abdominal aorta was clear, and the openings of the double renal arteries and the bifurcation of the common iliac arteries were defined. A 14 mm*10 mm Fogarty balloon catheter was placed into the sheath and inserted into the abdominal aorta below the opening of the renal artery, and the artery sheath and balloon catheter were fixed. The occlusion was observed after localization. The balloon was filled with diluted contrast agent, and abdominal aortic angiography was performed in parallel to show complete occlusion of abdominal aortic blood flow and smooth blood flow into the bilateral renal arteries. The focus was removed under hysteroscopy or laparoscopy immediately after the operation. During the operation, the balloon was filled intermittently to block the abdominal aorta blood flow. The balloon was filled for no more than 30 min at more than 10 min intervals . After tPatients with type III CSP in the UAE group were treated under DSA. Bilateral internal iliac artery and uterine artery angiography procedures were performed through unilateral femoral artery puncture. Catheters were inserted into the bilateral uterine arteries superselectively, and an appropriate amount of gelatine sponge granules was embolized into the peripheral vessels of the bilateral uterine artery. After embolization, the femoral artery puncture site was sutured and then pressure bandaged for 24 h. Hysteroscopy or laparoscopy combined with debridement was performed 1–3 days after uterine artery embolization. Figure The serum β-human chorionic gonadotropin (β-hCG) level was monitored weekly until it returned to a normal level . The serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and oestradiol (E2) were assessed six months after surgery.The clinical outcomes included intraoperative blood loss, time under DSA, body surface radiation dose, duration of surgery, postoperative serum β-hCG level, hospitalization expenses, duration of hospital stay, time to serum β-hCG normalization, and safety. The serum levels of FSH, LH, and E2 six months after surgery were also included.The assessment of adverse events included the occurrence of postoperative postembolization syndrome and decreased menstruation or amenorrhea six months after surgery. Postembolization syndrome was defined by the occurrence of pelvic pain, nausea, vomiting, fever, and perineal swelling . DecreasP value < 0.05 was considered statistically significant.The data analysis was conducted by IBM SPSS software version 26.0 . Continuous variables with normal distribution were reported as the mean ± standard deviation and compared by Student’s t tests between two groups. Continuous variables with nonnormal distributions were expressed as medians (interquartile ranges [IQR]) and compared by the Mann‒Whitney U test. Categorical variables are reported as numbers (%) and were compared by the chi-square test or Fisher’s exact test. A A total of 73 patients with a confirmed diagnosis of type III CSP between January 2020 and June 2022 were enrolled, of whom 5 were excluded. Finally, 68 patients were enrolled and randomized in a 1:1 ratio to the AABO group (34 patients) or the UAE group (34 patients) . The duration of surgery in the AABO group and UAE group was 35.5  min and 31.5  min, respectively (P = 0.300). The time under DSA in the AABO group was significantly lower than that in the UAE group ] s, P < 0.001). The body surface radiation dose in the AABO group was much lower than that in the UAE group .  and 10 IQR: 6.25, 20) mL, respectively . The duration of hospital stay was 2.5  and 4  days, respectively (P < 0.001). The hospitalization expenses were also significantly lower in the AABO group than in the UAE group . The time to serum β-hCG normalization in the AABO group was 28.9 ± 3.21 d, which was similar to that in the UAE group   U/L, 7.96  U/L and 5.63  pmol/L, respectively. In the UAE groups, the FSH, LH and E2 levels were 66.1  U/L, 8.07  U/L and 6.14  pmol/L, respectively. There were no statistically significant differences between the two groups.P < 0.001). No serious adverse effects occurred during treatment or follow-ups. None of the patients showed any sign of massive vaginal bleeding during or after surgery, and no hysterectomy was performed. In the AABO group, one patient experienced postembolization syndrome. In the UAE group, 16 patients had postembolization syndrome, including two patients with lower abdominal pain, 10 patients with hip pain, two patients with fever, and two patients with nausea and vomiting (Table The incidence of adverse events was 5.9% in the AABO group, which was less than that in the UAE group (58.8%, ng Table . DecreasCSP is considered a special type of ectopic pregnancy that must be terminated once diagnosed due to the increased risk of fatal bleeding. Studies have shown that prophylactic treatment can significantly reduce the risk of intraoperative haemorrhage in patients with type III CSP. However, there is no standardized treatment for type III CSP. In this study, 68 patients with type III CSP were treated with AABO or UAE as a prophylactic treatment. The results showed that the prophylactic AABO and UAE both are effective methods for patients with type III CSP, but AABO is safer and has fewer complications than UAE.Because the abnormally attached placenta has an extremely rich supply of blood vessels, bleeding during the operation is potentially fatal. In addition, due to difficult exposure of the operating field and the risk of massive bleeding, emergency haemostasis is difficult intraoperatively. Reducing the incidence of intraoperative bleeding and ensuring rapid haemostasis are essential to the successful rescue of placental attachment. Many studies have shown that patients with severe CSP III should undergo UAE to significantly decrease the risk of intraoperative haemorrhage , 21. In There was a previous study raising concerns for potential radiation injury in both the patient and foetus during AABO . HoweverIn this study, ovarian function was mainly assessed through sex hormone detection. Ovarian function in the patients in the two interventional treatment groups was comparable, and AABO presented a reduced risk of amenorrhea and infertility postoperatively. UAE is believed to cause ovarian ischaemia and decreased ovarian function. The cause for this may be uterine artery ovarian branch blockage or diffusion of embolization material into the ovarian vessels through the uterine artery, followed by partial vessel plugging . In addiIn our study, of the patients who underwent UAE, 10 complained of hip pain, and four suffered from decreased menstruation, with a total complication rate of 58.8%. Adverse events associated with UAE have been reported extensively. The overall incidence of postoperative complications ranged from 17.6 to 23% in the UAE pretreatment group . A recenDespite some significant findings that AABO is a reliable treatment method for type III CSP, especially when compared with UAE, there are still some limitations in this study. First, the small sample size is a major limitation. Large-scale clinical studies are needed to support our conclusions. Second, the sudden changes in total peripheral resistance caused by balloon occlusion and opening during abdominal aorta occlusion and reperfusion injury are likely to cause haemodynamic disorders, and markers of blood reperfusion injury should be evaluated after surgery. However, no further studies have been conducted on the haemodynamic changes of the two interventional methods due to lack of the patient acceptance.Both AABO and UAE are effective prophylactic methods for patients with type III CSP. In comparison to UAE, AABO is safe and has few complications. The effectiveness and safety of AABO should be confirmed in larger scale studies in the future.


Sixty genotypes with different seed coat colour and seed sizes were evaluated for seed longevity under both natural and accelerated ageing over seasons. The genotypes were grown during rabi, 2018, and summer, 2021, and freshly harvested seeds were used. For natural ageing, seeds were stored in a cloth bag in ambient condition and were removed at bimonthly intervals till 20 months. Accelerated ageing was carried out as per International Seed Testing Association (ISTA) guidelines. The germination percentage after natural and accelerated ageing over two seasons was determined. The correlation between two seasons of accelerated ageing and month-wise natural ageing was highly significant. The principal component analysis (PCA) using seed longevity grouped the majority of black genotypes into a separate cluster. Higher seed longevity was associated with black seed coat colour and small seed size. Microsatellite marker-based clustering also produced a separate cluster for majority of black genotypes and grouped the genotypes into a large number of clusters suggesting high diversity in the plant material. Two black seed coat colour genotypes, ACC No.369 and ACC No.39 consistently showed higher longevity under natural and both the years of accelerated ageing and serve as a source of alleles for higher seed longevity in soybean. Glycine max (L.) Merrill) is one of the major oilseed crops belonging to the family Fabaceae, subfamily Papilionoidea, and genus Glycine. The crop originated in Southeast Asia and was primarily domesticated by China around 1100 BC. Later it was introduced to several countries including India in the first century AD1. Soybean seeds are composed of 20% oil and 40% protein and contribute 70% to world’s protein and 28% to world’s oil consumption2. It is grown in an area of 119.0 million hectares with a production of 339.0 million metric tonnes worldwide and India produces 9.3 million metric tonnes from an area of 11.64 million hectares3. Due to its importance as a staple source of protein and oil for the growing population of the world, resources were allocated in the breeding effort to improve the grain yield with wide adaptability. A large number of varieties with high yield, early maturity, wide adaptability, and suitable for diverse agro-climatic conditions are developed in India and elsewhere4. Soybean seeds are very sensitive to production6, storage conditions8, seed handling, and mechanical damage11. The seed embryo and embryonic axis are located under a very thin seed coat and the surface of seed coat has presence of pores13. Hence, the seed viability and longevity are low15.Soybean  markers which are ubiquitously distributed within genomes are used to identify genetic diversity between genotypes in a germplasm collectionThe identification and development of high seed longevity genotypes in soybean is a requirement to be used as a source of germplasm for seed longevity in breeding. With this background, the present investigation was undertaken to elucidate the information on genetic and molecular diversity among the selected germplasm and identify genotypes with higher seed longevity through accelerated and natural ageing techniques and their association with seed coat colour and seed size.Rabi, 2018, and in Summer, 2021. Harvesting was done during January, 2019 and April, 2021, respectively for Rabi, 2018 and Summer, 2021. The seeds were immediately dried to a safe moisture content of 9% after manual threshing and the germination percent of the seeds were recorded before the start of the seed storage experiment.Sixty diverse genotypes including 46 yellow, three green , and nine black seed coat colour  were collected from author affiliated government institute, viz., All India Co-ordinated Research Project (AICRP)-Soybean scheme, Zonal Agricultural Research Station, GKVK, Bangalore, for the study and no permission is required for the collection. The genotypes, their name, and seed characteristics are given in Supplementary Table Rabi, 2018 experiment were tested for seed longevity under both natural and accelerated ageing, while the seeds harvested in Summer, 2021 were tested for longevity using accelerated ageing at AICRP-National Seed Project, University of Agricultural Sciences, Bangalore.The seeds obtained from 44 using between paper method. One hundred seeds in four replicates were randomly drawn from the cloth bag for each genotype at bimonthly intervals and kept for germination following between paper method. The seeds were kept in an incubator and the constant temperature of 30 °C and relative humidity of 90 ± 2% were maintained. The seeds were allowed to germinate and grow for 5 days. On the 5th day, the germination papers were removed from the incubator and the germination count was recorded following ISTA guidelines and expressed in percentage.Freshly harvested seeds were dried uniformly to 9% moisture content and stored in a cloth bag from February, 2019 to September, 2020 under ambient conditions (25–30 °C and 60–65% RH). At bimonthly intervals from the 8th month onwards till the 20th month after harvest, the seed samples were drawn from the stored cloth bags and used for testing the germination. The germination was recorded till the 20th month after harvest. The laboratory germination test was carried out as per the International Seed Testing Association (ISTA) guidelines39 for soybean. Four varieties, DSB 32, JS 95-60, Hardee, and DSB 33, were used as checks for the experiment. The box is filled with 40 ml of distilled water and it was sealed all around to maintain more than 95% relative humidity. The box was kept in an ageing chamber which maintain a constant temperature of 41 ± 0.3 °C for 72 h. After 72 h, the treated seeds were removed from the box, weighted, and kept for germination as mentioned above in four replications immediately (within an hour). The experiment was replicated twice.Freshly harvested seeds after drying to 9% moisture content were used for the study in both seasons. Forty-two gram seeds for each genotype were placed in an ageing box with a wire mesh screen by following the guidelines specified by ISTA45. The quality and quantity of DNA were estimated using 0.8% agarose gel electrophoresis with ethidium bromide staining and the DNA was uniformity diluted to 50 ng μl−1 and stored.The genotypes were grown in paper cups and the genomic DNA was isolated from young leaf tissues using cetyl trimethylammonium bromide (CTAB) methodFifteen microsatellite primer pairs were chosen for the study. A list of selected primers, primer sequence, and standardized annealing temperatures are given in Supplementary Table −1 ethidium bromide was used to visualize the marker alleles along with a 1000 bp ladder to score the fragment size.The isolated DNA was subjected to a polymerase chain reaction (PCR) using a reaction mixture of 10 μl and 38 reaction cycles. Three percent agarose gel with 0.5 μg mlThe germination percentage values were transformed using arcsine transformation to stabilize the variance. The analysis of variance (ANOVA) for the observed germination values for all the months and accelerated ageing was done by following a completely randomized design using SPSS software. PAST 4.03 software was used for plotting box plots and principal component analysis (PCA). Box plot was developed using the range, median, first, and third quartile values. PCA was undertaken based on scores of germination values from different storage months of natural ageing and accelerated ageing over two seasons. Eigen values and principal components were estimated using the germination values based on non-rotated loadings.46 based on their germination values in natural and accelerated ageing as given below,The correlation between two seasons accelerated ageing data and month-wise natural ageing data was determined using Pearson’s correlation coefficient. Further, the genotypes were classified into different longevity groupsμ’ and ‘σ’ are the mean and standard deviation of the overall germination of genotypes, respectively.Here, ‘The seed longevity ranks ‘SR 1’ and ‘SR 5’ reveals “highest” and “lowest” seed longevity, respectively. The genotypes which showed consistently higher ranking in both natural and accelerated ageing were considered as genotypes with higher longevity.47. Dissimilarity index was calculated using Euclidean distance with 1000 bootstraps and with this, hierarchical clustering of genotypes was done based on the unweighted pair group method with arithmetic mean (UPGMA) in DARwin 5.0 software.Marker scoring was done manually as 0/1 matrix. The presence of an allele was denoted as ‘1’ and absence as ‘0’. The band size matrix was used for the microsatellite genotypic data analysis. Allele frequency, genetic diversity and Polymorphism Information Content (PIC) were estimated using PowerMarker v 3.25 software. A number of different alleles (Na), number of effective alleles (Ne), Shannon’s information index (I), observed heterozygosity (Ho), expected heterozygosity (He), unbiased expected heterozygosity (uHe), and fixation index (F) was computed in GeneAlEx v 6.5The longevity of a seed is measured using its germination ability after a period of dry storage. A reduction in germination percentage of the genotypes was observed throughout natural storage under ambient conditions. The average germination % of genotypes after 8 months of ambient storage was 82.34 and it gradually declined to 3.35% after 20 months Table . By 14thThe analysis of variance suggested highly significant variation among genotypes for germination after ageing Table . The boxPCA is an alternative method of population structure analysis and it gives the explicit pattern of combination between the genotypes in the factor plane. In the PCA analysis of 60 soybean genotypes, the eigenvalues were higher for components one and two explaining 66.046 and 10.761% variance of the population, respectively Table , Fig. 2.Pearson’s correlation was performed to study the association between natural and accelerated ageing methods to test the reliability of accelerated ageing for screening the soybean genotypes for seed longevity. A significant positive correlation was observed between germination after natural ageing with germination after accelerated ageing in both the years Fig. . The cor., ACC No. 37, 109, LB-5, MAUS 71, and RKS 18 were consistently found in SR 2 rank of seed longevity. SR 1 and SR 2 rank classes are considered better for identifying genotypes for higher seed longevity. All the black seed coat genotypes, i.e., ACC Nos. 369, 39, 37, 109, and LB-5 were consistently found under either SR 1 or SR 2 rank. It is interesting to note that the seed size of the black seed coat colour genotypes which were grouped in higher longevity was small compared to other black seeded genotypes (Pune 14 and Pune 30) which were grouped in lower seed longevity rank (SR 3)  seed coat colour genotypes. The PCA scatter plot has been used to identify stress tolerant, high productivity genotypes in different crops60 and Kachare et al.57 wherein, the clusters obtained using phenotypic diversity was in accordance with the cluster found after performing SSR marker diversity.The molecular marker clustering also supports the results observed in PCA that the genotypes used for the present study are highly diverse at the genome level also. Like in PCA, one of the clusters formed using molecular markers contain majority of black seed coat colour genotypes. The clustering of genotypes using genotypic and phenotypic data are largely in agreement. The observations are in line with the finding made by Jeong et al.24 and Matera et al.38 also studied the association between natural and accelerated ageing in soybean and reported highly significant correlation between them in detecting seed longevity. The method has been widely used in crops like rice61, wheat62, chickpea33, Brassica63, soybean36, and palm64 for seed longevity. Accelerated ageing method is based on the principle of seed survival curve, that explains the relationship between seed viability and storage period. Accelerated ageing reduces the lag phase of a seed in declining its viability during storage37 by subjecting the seed to conditions that hasten the deterioration process. Such artificial ageing method is reliable, time saving, and useful for the quick assessment of segregants or genotypes in breeding. In the present study we have adapted both natural and accelerated ageing techniques to identify genotypes with higher seed longevity.The major objective of the present study is to identify the genotypes with higher seed longevity. After different duration of ageing of seeds under natural conditions and accelerated ageing, the seeds were tested for viability using germination percent. A significant positive correlation was found between natural ageing after 8th month till 20th month of storage under ambient conditions and the accelerated ageing method. Hosamani et al.65. In addition to these two black seed coat genotypes, other black seed genotypes, ACC No. 37, ACC No. 109, and LB-5 were found either in SR 1 or in SR 2 group. Seed longevity was also found to be associated with seed coat colour. The seed coat colour association with seed longevity observed in this study is in harmony with the earlier findings65. Kuchlan et al.13 reported that, black seeded genotypes have a minor gap between the seed coat and cotyledon, lesser pores on the surface of the seed coat, and greater lignin content in the seed coat, which makes them less susceptible to mechanical damage and deteriorative changes during ageing, than other seed coat colours. All the black seeded genotypes with high seed longevity had small seed size and lower 100 seed weight. The 100 seed weight ranged from 9.95 to 13.07 g. The other two black genotypes, Pune 14 and Pune 30 which did not show higher longevity had bigger seed size . It can be concluded that along with black seed coat colour, seed size are the determining factors for seed longevity in soybean30.Based on germination after natural and accelerated ageing methods, the genotypes were classified into different longevity rank groups. Majority of the genotypes were found in low seed longevity rank group of SR 3 and SR 4. Only a few genotypes were consistently found in the highest seed longevity group of SR 1. The two genotypes, i.e., ACC No. 369 and ACC No. 39 were frequently found in SR 1 group in both ageing methods and hence, the highest seed longevity among the genotypes. Both of them were found to be black seed coat colour genotypes. The black seed coat soybean genotypes were found to have relatively higher longevity1 plants have been identified using molecular and morphological markers and the F2 generation is being grown to develop RILs suitable for mapping seed longevity-related traits.Finally, in the study, contrasting accessions have been identified, which can be used for additional experiments to determine QTLs controlling the trait and also for breeding commercial lines. For this objective, the contrasting, yellow genotype with low longevity and black genotype with higher longevity have been crossed, and the true FSupplementary Information.