In the current study, six legume-cereal crop sequences, including faba bean, soybean, Egyptian clover, wheat, and maize were evaluated along two experimental rotations that ended up by planting the terminal maize crop. In addition, the effects of applying variable mineral nitrogen (MN) rates with and without the incorporation of farmyard manure (FYM) on the productive performance of maize and its NUE were tested. The field experiments were conducted in a no-tillage irrigated farming system in Northern Egypt, a location that is characterized by its arid, Mediterranean climate. Results revealed that increasing the legume component in the evaluated crop sequences, up to 75%, resulted in improved maize ear leaf area, 1000-grain weight, and harvest index, thus, a higher final grain yield, with the inclusion of Egyptian clover was slightly better than faba bean. Comparing the crop sequences with 50% legume contribution uncovered the positive effects of soybean preceding crop on the terminal maize crop. Substituting 25% of the applied MN with FYM resulted in similar maize yields to the application of the equivalent 100% MN rates. The fertilizer treatments significantly interacted with the crop sequences in determining the maize grain yield, where the highest legume crop contribution in the crop sequence (75%) equalized the effects of the different fertilizer treatments on maize grain yield. The integrated use of FYM with MN in maize fertilization improved the NUE compared to the application of MN alone. Comparing fertilization treatments with similar MN content, with and without FYM, revealed that the difference in NUE was attributed to the additional amount of FYM. In similar conditions to the current study, it is recommended to grow faba bean two years before maize, while Egyptian clover could be grown directly preceding maize growth, with frequent inclusion of soybean in the sequence, this could be combined with the application of an average of 200 kg MN ha The agricultural systems in Egypt and other developing countries are nowadays facing an unprecedented challenge in adequately feeding their continuously growing populations. Due to the extreme aridity of Egypt’s climate, agriculture is mainly dependent on irrigation ,2, thus,Among the efficient conventions and techniques utilized to achieve this goal is manipulating the order and frequency in which crops are grown, known as crop rotation . Crop roZea mays L.) is a principal annual cereal crop that occurs as a main component in the crop rotations in Egypt and other Mediterranean countries. Maize, a member of the Poaceae family, is the third-largest cereal crop produced worldwide after wheat and rice with a total production of 1148 million tons from a total harvested area of 197 million hectares in 2019 [−1 were produced, against 7.82 t ha−1 in 2009 [Maize  is the most important element required for improving maize growth and grain development ,20. It iIn the current study, it was hypothesized that manipulating the sequence of the preceding crops in rotation with maize would interact with the use of mineral and organic N sources in a way that uplifts the growth and productivity of the terminal maize crop, while positively altering the N use efficiency. The experiment was, therefore, set up to test, in particular, the effect of six legume-cereal crop sequences and the use of variable MN rates with and without the incorporation of farmyard manure (FYM) on the growth parameters and grain yield of maize grown under a no-tillage farming system in Northern Egypt.p < 0.01) influence on all the studied parameters except plant height, ear height, and leaf area index (p > 0.05) in the two growing seasons. Moreover, all the investigated parameters were highly significantly (p < 0.01) affected by the fertilization treatments except, plant height and leaf area index that were significantly (p < 0.05) affected, and ear height that was non-significantly affected in both seasons. Meanwhile, the leaf area index (p < 0.05), ear grain weight, and grain yield (p < 0.01) were significantly variable as affected by the two-way interaction between the crop sequence and fertilization treatment. The main effects of the two studied factors will be presented and discussed only when their interaction is not significant.Mean squares presented in Means of the studied parameters as affected by the crop sequences are illustrated in 2, for 2018 and 2019, respectively, against only 618.20 and 657.00 cm2 for CS1 during the two respective seasons. Noticeably, CS2 was also characterized by low ear leaf area compared to the other tested crop sequences.The highest significant ear leaf area C was repThere were significant variations in 1000-grain weight among all crop sequences D. Data rRegarding the HI, means E showed It was clear from the means presented in 2, in addition to FT6 during the second season (794.26 cm2). Although FT1 and FT2 resulted in intermediate ear leaf area, their reduction by 25% in addition to FMY (FT4 and FT5) resulted in significantly smaller ear leaf area during both seasons, with the lowest significant values reported for FT4, amounting to 592.00 and 624.52 cm2 for 2018 and 2019, respectively. These values represented a 23.05 and 21.98% decrease in the ear leaf area than the highest significant values reported for FT3, during the two respective seasons (Ear height was non-significantly variable among the tested fertilization treatments and ranged from 140.10 cm (FT4) to 149.90 cm (FT6), during 2018, and from 140.40 cm (FT4) to 148.72 cm (FT3) during 2019 B. The hi seasons C.Data in Harvest index (%) of maize varied significantly among all the tested fertilization treatments during both seasons E. The hiLeaf area index (LAI) means presented in A similar trend was observed for the ear grain weight, as affected by the interaction between the crop sequence and fertilization treatment . While C−1. However, more pronounced variations among fertilization treatments were observed in 2019. It was observed that the lowest significant grain yields under CS1, CS2, and CS3 accompanied the application of FT4, amounting to 5.71, 5.91, and 6.02 t ha−1, for the three respective crop sequences. Nonetheless, at CS4, CS5, and CS6, FT3, FT5, and FT6 produced the highest significant grain yield.Means of maize grain yield as affected by the interaction between the evaluated crop sequences and fertilization treatments are presented in Data of the NUE for the evaluated fertilizer treatments during 2018 and 2019 are illustrated in 2 fixation, and (3) maintain the essential diversification to the cropping system (as break crops) that would uplift the productivity of the system, compared to cereal-cereal rotations [The effect of variable legume-cereal crop sequences on the yielding potential of the terminal maize crop in a rotation is well documented by several researchers ,16,17, yotations . Based ootations , soybeanotations , and Egyotations .2 through their symbiotic association with the rhizobium bacteria allows them to play a vital role in the crop sequence. Important nutrients (like N) could be released through the mineralization of the preceding legume crop residues, thus, creating favorable conditions for the growth and activity of the useful soil microorganisms [In the current study, increasing the legume component in the evaluated crop sequences, up to 75% (CS5 and CS6), resulted in improved maize growth parameters and, thus, higher final grain yield. This is most probably attributed to improving the soil environment, through the decomposition of the legume crop residues . The abirganisms , and balrganisms . In addirganisms ,46. Furtrganisms . MeanwhiAmong the fertility-building plants, clovers are known for their distinguished positive impact on the succeeding cereal crops. This was reported for red, white, crimson, and Egyptian clovers ,49. Fora2 fixation, but also include reducing the incidence of grassy weeds, pests, and diseases [The reported benefits of including faba bean in crop sequences with cereals, are not limited to Ndiseases ,51. Despdiseases , the posdiseases  reporteddiseases , highligdiseases , who docdiseases .The choice of the break crops to be included in a particular crop sequence should be juxtaposed against the expected profitability from the whole rotation . Thus, fThe ideal N management improves soil fertility, optimizes crop productivity , and incEven though the highest MN rate resulted in the highest maize growth attributes and grain yield, this rate is no more recommended as it is associated with the production of the tallest significant maize plants, making the crop more prone to lodging . Nonethe−1, as the appropriate MN level for improved grain filling process and lodging resistance in maize. This supports the current findings, that FT5 and FT6, containing 188 and 214 kg ha−1 MN, respectively, were the most appropriate MN rates, when compensated with FYM.Applying soil organic amendments is nowadays thought of as one of the efficient solutions to the above-mentioned obstacles. However, controversial results were reported in previous studies concerning the effect of incorporation of organic amendments in maize fertilization scheme on grain yield and yield components. While, , reporte2 fixation by legumes. It could be, thus, concluded that maize rotation with legume crops can generate enough N in the soil to meet a large part of the demands of maize, thus, reducing the required amount of N from external sources [In the current study, the fertilizer treatment significantly interacted with the crop sequence in determining the maize grain yield, where the highest legume crop contribution in the crop sequence (75%) equalized the effects of the different fertilizer treatments on maize grain yield. This suggests that the high legume component in the crop sequence compensated for the low N content in the fertilization treatment, mostly because of the biological N sources . However sources , its bio sources , and the sources . A large sources , for vet−1  resulted in a lower total N content than the application of the 100% MN rates , yet a higher NUE and grain yield. Moreover, results indicated that, despite similar MN content, yet higher total N content, FT6 was superior to FT1 in improving NUE, suggesting that this difference in NUE was attributed to the additional amount of FYM in the FT6. This implies that the N from organic sources does not have a similar detrimental effect on NUE as the same amount of N from mineral sources.Although high N rates are needed for achieving high yields, they are associated with reduced NUE ,74,75, mA field experiment was carried out over two periods of time; from 2016 to 2018, and from 2017 to 2019 at the Agricultural Research Station, Crop Science Department, Alexandria University, located at 31.22° N, 29.94° E. The experimental location was characterized by its Mediterranean climate, average monthly temperature (°C), and humidity (%) for the two summer seasons (2018 and 2019), where the terminal maize crop was planted, are presented in A factorial layout of treatments in a three-replicated randomized complete block design (RCBD) was adopted for the field experiments, to investigate the effect of six crop sequences and six fertilization treatments on the growth and productivity of the terminal maize crop. Six crop sequences were investigated in the current study, in which three different crops were grown along the experimental periods, that ended up with growing the terminal maize crop. The different crops and cultivars included in the six investigated sequences were: Maize (Giza 168), faba bean (Giza 843), soybean (Giza 111), wheat (Gemmeza 9), Egyptian clover . The studied crop sequences in both experimental periods are presented in −1, FT2 = 250 kg N ha−1, FT3 = 285 kg N ha−1 (the recommended N rate for maize growing in the region), FT4 = 161 kg N ha−1 + 9.6 t ha−1 FYM, FT5 = 188 kg N ha−1 + 9.6 t ha−1 FYM, FT6 = 214 kg N ha−1 + 9.6 t ha−1 FYM. The mineral nitrogen (MN) was applied in the form of ammonium nitrate (33.5% N). The FYM was added at a rate of 9.6 t ha−1, containing 3.60 and 3.21 kg ton−1 nitrogen and phosphorus respectively. Thus, the added amount of FYM (9.6 t ha−1) provided around 35 kg organic N ha−1. FYM was manually incorporated into the soil 3 weeks before sowing of the terminal maize crop. The used FYM had a pH value of 6.9, EC value of 4.3, and 17.6, 1.18, 1.07%, for total C, N, and P, respectively, in addition to 14.92 C/N ratio.Six fertilization treatments were investigated in the current study and included three mineral nitrogen (MN) full rates. The other three fertilization treatments included 25% less MN rates than the previous three treatments plus a fixed amount of organic farmyard manure (FYM). Thus, the investigated fertilization treatments were as follows; FT1 = 215 kg N haThe first experimental rotation was initiated in winter 2016/2017 and terminated in summer 2018, while the second experimental rotation started in winter 2017/2018 and ended in summer 2019. Both experimental rotations ended up by planting the terminal maize crop. A fixed layout was maintained since the start of the experiment and applied to both experimental rotations. A schematic diagram of the evaluated crop sequences in the two experimental rotations illustrating the sowing order and dates of the different crop components is given in 2. All crops were planted following the recommendations in the region regarding sowing method and seeding rate. Wheat was drilled on the upper side of the raised wide bed, in rows 30 cm apart, with a 144 kg ha−1 seeding rate. Two grains of Maize were sown in hills (30 cm apart) and thinned to one plant per hill 14 days after sowing to maintain the recommended plant density . Egyptian clover was broadcasted with a 48 kg ha−1 seeding rate. Faba bean and soybean were sown in hills, that were 30 and 20 cm apart, respectively, and in rows (30 cm spaced), and thinned to two plants per hill, 14 days after sowing for both crops. The used seeding rate was 96 and 72 kg ha−1, for faba bean and soybean, respectively. Prior to sowing, seeds of faba bean, soybean, and Egyptian clover were inoculated with the appropriate Rhizobium spp. to enhance biological N2 fixation. The specific strains, namely; Rhizobium leguminosarum bv. viciae, Bradyrhizobium japonicum, and Rhizobium trifolii were used to inoculate faba bean, soybean, and Egyptian clover seeds, respectively. A zero-tillage strategy was adopted along with the two experimental rotations, to avoid any soil disturbance and keep the preceding crop residues in the soil. The experimental plot comprised four raised wide beds, each was 3 m long and 1.2 m wide, resulting in a total plot area of 14.4 m2O5 ha−1, applied as calcium monophosphate (15.5% P2O5), was added to the soil with seedbed preparation. Meanwhile, nitrogen in the form of ammonium nitrate (33.5% N), was added as 36 kg N ha−1 for faba bean, Egyptian clover, and soybean, 144 kg N ha−1 for wheat, and 285 kg N ha−1 for maize, which was split into two equal applications, added with the first and second irrigations. The terminal maize crop received the recommended phosphorus rates, in addition to the tested fertilizer applications as a source of organic and mineral N. To avoid induced drought stress, surface irrigation was scheduled on 14- and 7-day intervals during the winter and summer seasons, respectively. No serious incidence of pests or diseases was observed along the experimental periods, and hand weeding was always practiced with all the crop components.All component crops in the evaluated crop sequences were fertilized upon the recent recommendations of the Egyptian Ministry of Agriculture and Land Reclamation. The recommended phosphorus rate of 36 kg P2), and leaf area index were determined for five randomly chosen plants from each experimental plot. At harvesting, the stalks of the maize plants on the inner two wide beds for each plot were manually cut with a sickle directly above ground level, then the total biological yield was immediately weighed in the field. After removing the ears from the plants, they were separately shelled and grain yield was weighed as kg plot−1, in addition, the ear grain weight (g) was calculated as an average of five random ears from each plot. The 1000-grain weight (g) was determined for three random grain samples taken from each plot. Harvest index (HI) was calculated as grain yield divided by total biological yield and expressed as a percentage.At grain full maturation and prior to harvesting of the maize terminal crop, plant height (cm), ear height (cm), ear leaf area (cmNUE) was calculated after [fG is the grain yield of the fertilized plot, and aMN is the total amount of applied mineral N at each fertilization treatment.Nitrogen use efficiency (ed after  as the gp) were analyzed according to the following model:µ is the overall mean, iR is the replication , jCS is the crop sequence effect , kFT is the fertilization treatment effect , (CS × FT)ij is the effect of the interaction between the crop sequence and fertilization treatment, and ijke is the experimental error.The crop sequences, fertilizer treatments, and their interaction were tested for significance using Proc Mixed of SAS 9.4  after 8. Only rep < 0.05.Prior to the statistical analysis of the data, the harvest index was arcsine transformed and expressed as a percentage. Means were compared using the least significant difference (L.S.D) procedure, with significances declared at −1 in addition to FYM.The present research highlighted opportunities for the improvement of maize productivity when grown in a no-tillage farming system, in an arid Mediterranean region, through manipulating the preceding crop sequence and N fertilizer input. Increasing the legume component in the evaluated crop sequences, led to higher maize grain yields, with the inclusion of Egyptian clover being slightly better than faba bean. Especial emphasis was made on the importance of including soybean in the crop sequence preceding the terminal maize crop. Substituting 25% of the applied MN with FYM resulted in similar maize yields to the application of the equivalent 100% MN rates, under high MN rates, thus, a minimum MN rate is always required to achieve a reasonable amount of maize yield. The highest legume crop contribution in the crop sequence (75%) equalized the effects of the different fertilizer treatments on maize grain yield. The integrated use of FYM with MN in maize fertilization improved the NUE compared to the application of MN alone, suggesting that the N from organic sources does not have a similar detrimental effect on NUE as the same amount of N from mineral sources. In similar conditions to the current study, it is recommended to grow faba bean two years before maize, while Egyptian clover could be grown directly preceding to maize, with a frequent inclusion of soybean in the sequence and this could be combined with the application of an average of 200 kg MN ha


The rise in energy drink (ED) intake in the general population and athletes has been achieved with smart and effective marketing strategies. There is a robust base of evidence showing that adolescents are the main consumers of EDs. The prevalence of ED usage in this group ranges from 52% to 68%, whilst in adults is estimated at 32%. The compositions of EDs vary widely. Caffeine content can range from 75 to 240 mg, whereas the average taurine quantity is 342.28 mg/100 mL. Unfortunately, exact amounts of the other ED elements are often not disclosed by manufacturers. Caffeine and taurine in doses 3–6 mg/kg and 1–6 g, respectively, appear to be the main ergogenic elements. However, additive or synergic properties between them seem to be implausible. Because of non-unified protocol design, presented studies show inconsistency between ED ingestion and improved physical performance. Potential side effects caused by abusive consumption or missed contraindications are the aspects that are the most often overlooked by consumers and not fully elucidated by ED producers. In this review, the authors aimed to present the latest scientific information on ED components and their possible impact on improving physical performance as well as to bring emphasis to the danger of inordinate consumption. Energy drinks (EDs) are widely available beverages containing caffeine, taurine, glucose, and other ingredients and are said to improve physical or cognitive performance and should not be confused with isotonic drinks or sport drinks . EDs firn = 52,016) recorded a 68% lifetime prevalence of ED use among adolescents (aged 10–18 years old). Among the declared users, 12% were “high chronic” consumers with an average consumption of 7 litres per month. In adults, the prevalence of consumption was 30%, and it 18% in children (aged 6–10 years old) [n = 8210), where 62% of adolescents reported consuming EDs at least once in the previous year [n = 6902) reported that 61.7% of German adolescents between 9–19 years of age had used ED at least once. Degirmenci et al. [n = 31,081). Presented data show that a young population, mainly between 9–19 years old, constitutes the biggest pool of ED consumers, with the prevalence of lifetime use ranged from 52% to 68%.In 2013, a study conducted in 16 European countries , Petróczi et al. [n = 707, mean age 14.3) consumed EDs. Most often, they drank 250 mL daily. Moreover, the same authors noted that adolescents who practiced sports were more willing to drink EDs, and 28% of them claimed that EDs gave them a boost of energy [The prevalence of ED consumption among athletes, especially professional sportspersons, is limited and varies across studies. Cross-sectional analysis of survey data of a cohort-study of 2287 young adults (mean age 25.3 years) demonstrated that men who spend more time engaged in physical activity had higher ED consumption and were more likely to use muscle-enhancing substances [f energy . On the f energy  in a US f energy  and studf energy , supportThe high prevalence of ED consumption among the young population may result from a well-conceived marketing strategy that persuades them to purchase and consume EDs . One of As EDs have risen in popularity over the years, particularly among young people, considerable debate should take place over the validity and safety of their intake. This review is an attempt to describe the present state of knowledge about the ED components and their biochemical and ergogenic properties as well as to emphasize to the danger of the short- and long-term adverse effects of inordinate consumption.ED compositions vary widely, and manufacturers often do not disclose the exact quantities of particular ingredients. Furthermore, ED companies do not distinguish which component, alone or in combination, explains the potential positive benefits of the drink ,3. Howev1R and A2aR in the brain can modulate proinflammatory cytokine production. This property might interfere with the inflammation process after physical exercises, when the levels of cytokines, mainly TNF-α, IL-1β, IL-6, and IL-10, are elevated [2+ release into the intracellular space. This effect is possibly achieved by caffeine acting as an antagonist of the adenosine receptor subtype A1 on the skeletal muscle membrane or/and attachment to ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) [2+, increased myofibrillar Ca2+ sensitivity, improved SR Ca2+ permeability, and decreased activity of the SR Ca2 pump are attained, ultimately resulting in modified muscle performance [The ergogenic effects of caffeine are connected to its biological activity in the central nervous system and in the peripheral organs. Being a nonselective adenosine receptor antagonist, caffeine acts as central nervous system stimulant via the inhibition of the sleep-promoting adenosine effect that results in the release of dopamine, serotonin, and norepinephrine in the brain . The secelevated . The efflum (SR) ,24. Besiformance . Furtherformance . These eformance ,27. Howeformance . Systemaformance . In theiformance  demonstrformance  showed nformance . These rThe next key component of ED is taurine, a sulphur-containing amino acid widely distributed in tissues and naturally produced by the human body . Dietary2+-ATPase (SERCA) function, either via an augmentation in its maximum SR Ca2+ pumping rate or its affinity for Ca2+, which finally led to an increase in the rate of Ca2+ accumulation by the SR. However, the maximum Ca2+ content produced by the SR was not affected. SR Ca2+ accumulation was not changed when the intracellular taurine concentration was higher than physiological range, but lowering the taurine level below its normal range resulted in significantly decreased SR Ca2+ content. This observation led authors to speculate that such a decrease in the SR Ca2+ level would adversely affect muscle function because of its slower relaxation. Second, intensive activity of the skeletal muscle cells leads to an increased production of reactive oxygen species (ROS), which contribute to contractile proteins oxidation, including the inhibition of SERCA [2+ homeostasis [The physiological activity of taurine is widespread and includes the modulation of mitochondria function, bile acid conjugation, the regulation of cellular calcium levels, and cytoprotective properties . This amof SERCA . Taurineof SERCA . This prof SERCA ,43. Besieostasis . The taup = 0.004). Moreover, the taurine dose did not have an impact on its ergogenic effect and could be as low as 1 g or as high as 6 g. The differences between sexes were not investigated [Whilst the impact of caffeine on physical performance is well-established, the evidence of taurine efficacy is limited. A systemic review based on 10 studies with 128 volunteers, showed that an isolated dose of taurine in varying amounts (1–6 g), regardless of acute or chronic supplementation, has the potential to improve overall endurance performance , Jeffries et al. [On the other hand, in their double blind, cross-over study , endurance exercise tests , jumping , and sport-specific actions , but it did not any improved physical performance in sprinting (ES = 0.14); p = 0.06). Interestingly, a significant association between taurine dosage and increased physical performance (p = 0.04) was found, but not between physical performance and caffeine dosage (p = 0.21). Unfortunately, the EDs contained other potentially ergogenic elements, which may have influenced the final results, but were not included in the comparison.Due to the popularity of energy drinks and their potential ergogenic effect, many studies undertake the topic of correlation between ED ingestion and improved physical performance. In their meta-analysis of 34 studies published between 1998 and 2015 with 653 subjects, Souza et al.  demonstrp = 0.016). A total of 78% of the participants performed better after ED ingestion. The amount of caffeine ingested was in the range of 1.5–3.9 mg/kg (average 2.9 mg/kg), and the relation between caffeine intake and the 5-km running time demonstrated a weak correlation. The average amount of consumed taurine was 28 mg/kg, but the association between its dose and physical outcomes was not investigated.Prins et al.  conductep = 0.015). This effect was maintained even after an exhaustive bout of high-force contractions. However, the strength change for the uncaffeinated ED was not significantly different from the caffeinated ED, suggesting that caffeine was not the sole ergogenic agent in the used beverages [A double-blind study by Collier was performed on 15 physically active students who were familiar with resistance training. Every participant served as their own controls and were tested across three trials, each separated by 7 days, ingesting either a caffeinated, uncaffeinated, or a placebo beverage. The amount of caffeine received from the caffeinated ED was 5 mg/kg, whereas the exact taurine dose was not disclosed. The results of the study presented a 5% improvement in the maximum voluntary isometric contraction strength of the knee extensors after the ingestion of a caffeinated ED compared to the placebo (everages . p < 0.05) but was not sufficient to affect the larger muscle groups of the lower legs . Moreover, a comparison of gender differences implied that only women improved their forehand stroke velocity after ED intake, while the results of a vertical jump test were similar between the sexes. The amount of caffeine in the EDs was constant and amounted about 240 mg, and the men were over 23% heavier than the women. Thus, the men received an average dose of 2.8 mg/kg, whereas the women ingested an average dose of nearly 4.0 mg/kg. The taurine dose was not disclosed, but other ingredients were also present. Presumably, the variabilities in body mass, body composition between sexes, and caffeine distribution in various tissues in proportion to their water content could cause the abovementioned differences.Research performed by Jacobson et al.  on 36 php = 0.03) in both hands, running pace at high intensity (p = 0.02), and the number of sprints (p = 0.05) during the simulated match. There were tendencies for increased maximal and mean running velocity tests and higher percentage of points won on service with the caffeinated ED, although the differences did not reach statistical significance. However, during the serving test, no improvements were found in ball velocity and total distance. The limitation of the study was lack the of placebo beverage without potentially active components.Gallo-Salazar et al.  conductep = 0.25, p = 0.006, respectively). However, the authors concluded that the acute ingestion of the ED did not improve the physical performance of the female volleyball players. It is worth mentioning this was the second study that presented that smaller muscle groups tend to be more sensitive to ED ingredients than larger muscles [On the other hand, a number of published studies failed to report statistically significant ED influence on physical performance. A double-blind, crossover study of Fernández-Campos et al.  conducte muscles .p < 0.157) and VO2max (p < 0.154) compared to the placebo. However, this study presented the effects of EDs on an untrained population in comparison to amateurishly or professionally trained participants in previous studies. Moreover, a limitation of the study was single-blinded design.Al-Fares et al.  conductePresented investigations show that EDs have the potential to improve physical performance. However, the mechanism of this enhancement and main substance responsible for that effect maintains unclear. A meta-analysis of 34 studies highlighted a significant association between taurine dosage and better performance . A similSummarizing the results of this part, adult athletes without comorbidities who are willing to improve their physical performance may consider using isolated caffeine in a minimal 3 mg/kg dosage ingested 30–60 min before exercise ,29,30,31The summary of the studies describing the impacts of EDs on physical performance is shown in The popularity of EDs, especially among adolescents and young adults, has brought many questions and concerns about their overall safety and health in the scientific community. Tempting slogans and sponsorship in extreme sports and e-Sports are cleverly used by ED campaigns to promote their products without the necessity of mentioning their side effects ,19,20. UThe short- and long-term effects of the ED consumption are arguable, although the short-term effects appear to be dominant. The most common adverse effects are associated with the cardiovascular and neurological systems . A meta-According to various reports, EDs can cause all kinds of arrhythmias, with occurring atrial fibrillation (AF) the most often . QT prolA significant connection between ED consumption and mental health problems among adolescents has been found in several studies. They were associated with a greater risk of moderate and serious levels of psychological distress, suicidal thoughts, and suicide attempts . SimilarThe reported metabolic impact of EDs is contradictory. A recent prospective study with a two-year follow-up duration (measurements were taken at 20-years and at 22-years) did not find compelling evidence between frequency of ED-intake, BMI, metabolic syndrome, blood pressure, fasting glucose, triglycerides, and HDL . HoweverIn addition, a recent study on 129,809 Korean adolescents presented that frequent ED consumption was associated with allergic diseases, such as asthma, allergic rhinitis, and atopic dermatitis . The lasBeyond the previously mentioned alarming effects of ED consumption, there are extra concerns for athletes. EDs induce diuresis and slow fluid absorption, which may exert a negative influence in particularly during hot weather and during ultra-resistance sports competitions like marathons ,51,64. IUnclear pharmacology and interactions between various ingredients found in these drinks put children at risk of dangerous consequences . ConsideThe summary of the studies describing the adverse effects of energy drink consumption is shown in According to this review, high-quality studies are undeniably needed. One of the main aspects that should be taken into account is evidence suggesting a potential interaction between caffeine and taurine. This evaluation would answer the question whether using these supplements together is beneficial and would mark a direction for subsequent studies of ED ingredients. Moreover, knowledge of these interactions would make side effects more predictable in case of inordinate usage. Due to different protocol design, there are many incompatible studies about the correlation between ED usage and exercise performance in athletes. It is still uncertain if ED supplementation should be recommended and what doses of particular ingredients should be added to trigger optimal improvement among athletes. Only the unification of protocol design will significantly answer the question of whether EDs can effectively help in improving physical performance.


Moringa oleifera trees in this type of soil is of special interest given the increasing demand for every part of this tree. Several studies have focused on the production of its leaves as an herbaceous plant and not as a tree, but there has not been extensive research on its pods, seeds, and fixed oil production. In this sense, in this study, we provide an assessment of the use of fertilizers, vermicompost and NPK , in order to improve pods, seeds, and fixed oil contents, as indicators of the quality of the production of the Moringa oleifera trees in calcareous soil conditions. In this experiment, it was observed that all parameters and the yield of pods, seeds, and fixed oil of the Moringa oleifera tree were significantly improved by increasing the level of vermicompost and using NPK fertilization and combination treatments in both seasons of the study. The combination treatments of 10 and 20 ton ha−1 vermicompost plus NPK control produced the highest percentage of oleic acid with insignificant differences between them.A shortages of soil nutrients resources and a lack of accessibility to them especially in calcareous soil are considered some of the main factors that limit plant production. In particular, the cultivation of the  Moringa oleifera is a small or medium-sized tree, about 10 m high. It is commonly known as a horseradish or drumstick tree and all its parts are useful. It belongs to the Moringaceae family, which consists of the single genus Moringa, comprised of 10–14 species. The best-known species is Moringa oleifera, which is indigenous to northwest India and widely cultivated in the Philippines, Thailand, Malaysia, Pakistan, and other tropical and subtropical areas in Central Asia, America, and Africa [Moringa oleifera tree is nutritional and rich in vitamins and minerals [Moringa oleifera tree is very useful in animal feed, alley cropping, household cleaning agents, biogas, medicine, green manure, ornamental plants, gum and water purification [Moringa oleifera tree such as the leaves, inflorescences, seeds, and roots [d Africa , and is minerals . In addiminerals . This oification . Moreovend roots .Moringa oleifera tree in slightly acidic to neutral well-drained loam to clay loam soils [Moringa oleifera tree and enhance its ability to give a healthy plant [Soil is the main support for plants in agriculture, due to its contribution of nutrients through the soil solution and its function of providing physical support for plants. It is preferable to culture the am soils . Furtherhy plant  that wilHowever, agricultural production in calcareous soils faces many difficulties due to a high level of calcium carbonate; high infiltration rate; low water holding capacity; low organic matter and clay content, poor structure, low cation-exchange capacity, surface crusting and cracking; loss of nutrients via leaching or deep percolation; high pH with a nutritional imbalance between elements such as potassium, magnesium, and calcium; and low availability of nutrients, in particularly phosphorous, iron, and micronutrients. These difficulties can result in failure to obtain the desired plant growth and yield ,10,11,12Moringa oleifera trees. Under these conditions, foliar fertilization is important as a part of fertilization programs for the Moringa oleifera trees, especially using nanofertilizers in a nanoscale range of 1–100 nm that allows them to penetrate the plant tissues. In this field, foliar application of nano-NPK fertilizers reduces soil pollution and obviates the interaction of nutrients with water, microorganisms, and calcareous soil, and therefore reduces the amount of nutrients used to below recommended levels [Nitrogen is the main element to provide plants with the required amino acid and protein in plant. Under the alkaline conditions of calcareous soil, the rate of N transformation increases and the efficiency of N use by plants can be influenced. Therefore, minimizing ammonia volatilization and leaching of N presents the proper N management for fertilization in calcareous soil ,14. Phosd levels . In theid levels , Sheykhbd levels , Bozorgid levels , and Hagd levels , the autVermicompost is one of the most favorable non-chemical plant nutritional sources. It can be produced through vermicomposting of organic material by earthworms, which can consume a wide range of organic residues. It has a positive influence on the physical and chemical structure of soil as well as plant growth  by improPseudomonas striata [(i) Vermicompost increases the availability of plant nutrients in soil by adding N, P, K, as well as other micro- and macro-nutrients during the process of mineralization of organic matter. Vermicompost contains a larger group of soil-friendly fungi, bacteria and actinomycetes ,26, such striata , as plan striata . Herenci striata  reported striata , which r striata .Zea mays.(ii) Vermicompost stimulates growth-promoting hormones such as auxins, gibberellins, and cytokinins that are produced by microorganisms in vermicompost ,35, whic(iii) Vermicompost provides biological control of plant and soil pathogens. Moringa oleifera trees in this study. Furthermore, organic fertilizers are suitable for achieving the objectives of sustainable agriculture as noted by many researchers, such as Arancon et al. [(iv) Soil health is improved by the organic matter content in vermicompost. Generally, the addition of vermicompost as an organic source to calcareous soil increases the nutrient cycling; retains soil water, soil structure, and biological processes ,39,40,41n et al. , Joshi an et al. , Salehi n et al. , Madahi n et al. , and Oftn et al.  who studn et al.  reportedMoringa oleifera trees, several studies, such as those by Sánchez et al. [Moringa oleifera tree as an herbaceous plant, not a tree, and therefore they have focused on its leaf production under normal conditions. However, there has not been exhaustive research on the mature tree and its pods, seeds, and fixed oil production. Consequently, this study was carried out to assess the response of the Moringa oleifera tree to different fertilization rates using vermicompost and NPK , in order to determine, under calcareous soil conditions, the rate that corresponds to optimum pod, seed, and fixed oil yield, as well as the percentage of oleic fatty acid.Despite the enormous potential of the z et al. , Isaiah z et al. , and Danz et al. , have trMoringa oleifera trees were significantly affected by different treatments of vermicompost, NPK, and combination treatments ; the weight of mature pods (10.17 and 9.76 g), and the maximum mean values of number of pods per tree (164.6 and 176.1) in the first and second seasons respectively, were reduced by adding 50 ton ha−1 vermicompost across all NPK levels. In addition, when nano-NPK was used the maximum mean values of the number of pods per inflorescence (2.89 and 3.00), the number of pods per tree (140.6 and 147.6), and the weight of mature pods (9.27 and 9.03 g) were obtained across all vermicompost levels, in the first and second seasons, respectively. The interaction between vermicompost and NPK treatments was highly significant. Moreover, the 60 ton ha−1 vermicompost plus 2 gL−1 nano-NPK treatment resulted in the maximum highly significant mean values of the number of pods per inflorescence (3.53 and 3.69) and the weight of mature pods (11.22 and 10.93 g), in the first and second seasons, respectively; the maximum mean values of the number of pods per tree  were produced with the 50 ton ha−1 vermicompost plus 2 gL−1 nano-NPK combination treatment  and per hectare  in the first and second seasons, respectively. On the other hand, by using the 50 or 60 ton ha−1 vermicompost treatments, insignificant differences were found in the mean values of mature pods per tree and per hectare, in both seasons. Regarding NPK fertilizer, the 2 gL−1 Nano-NPK treatment resulted in the maximum mean values of yield of mature pods per tree (1425 and 1454 g tree−1) and per hectare  in the first and second seasons, respectively, across all vermicompost levels. The interaction between vermicompost and NPK treatments was high significant. Moreover, the 50 ton ha−1 vermicompost plus 2 gL−1 nano-NPK treatment produced the maximum mean values of yields of mature pods per tree (2616 and 2750 g tree−1) and per hectare  in the first and second seasons, respectively.The results of the highly significant effects of different treatment levels of vermicompost, NPK, and their combinations on the yield of mature pods of the −1 vermicompost treatment as compared with the other vermicompost treatments, including the control. The same situation was observed for the 2 gL−1 nano-NPK treatment which recorded the maximum mean values of the number of seeds per pod (19.3 and 19.5) and weight of mature seed weight (0.208 and 0.210 g) as compared with the treatments of 2 gL−1 and the NPK control in both seasons. Regarding the combination treatments, the 60 ton ha−1 vermicompost plus 2 gL−1 Nano-NPK treatment produced the significant maximum mean values of the number of seeds per pod (22.8 and 23.0) and weight of mature seed weight (0.267 and 0.270 g) in the first and second seasons, respectively  and mature seeds (0.228 and 0.231 g) in the first and second seasons, respectively, were produced by the 60 ton haectively .Moringa oleifera seeds per tree and per hectare (−1) and per hectare (7777 kg ha−1) was maximum with 50 ton ha−1 vermicompost treatment. In the second season, the 60 ton ha−1 vermicompost treatment produced the highest mean yield of mature seeds per tree (868 g tree−1) and per hectare (8678 kg ha−1) as compared with the other vermicompost treatments including the control. For both seasons, the differences between the mean yield of mature seeds per tree and per hectare with the 50 and 60 ton ha−1 vermicompost treatments were insignificant. Regarding the use of NPK, it was observed that spraying 2 gL−1 nano-NPK gave the maximum mean values of yields of mature seeds per tree (676 and 737 g tree−1) and per hectare (6759 and 7370 kg ha−1), in the first and second seasons, respectively, as compared with the 2 gL−1 NPK treatment and NPK control. Regarding the combination treatments, the 50 ton ha−1 vermicompost plus 2 gL−1 nano-NPK treatment gave the maximum yields of mature seeds yield per tree (1350 and 1482 g tree−1) and per hectare  in the first and second seasons of the study, respectively.As compared with the control treatment, different levels of vermicompost, NPK, and their combinations showed significant improvements in the yield of mature  hectare . In the Moringa oleifera seeds was significantly affected by most levels of applied vermicompost, NPK, and their combinations treatments in both seasons (Moringa oleifera seeds in this study was decreased by increasing the vermicompost level. The vermicompost control had the maximum fixed oil percentage (35.89 and 36.50%). Insignificant differences were detected between the control and the 10 ton ha−1 vermicompost treatment, in both seasons. The same situation was found for the effect of NPK on fixed oil percentage as the NPK control gave the maximum highly significant percentage of fixed oil (34.53 and 33.83%) as compared with 2 gL−1 NPK or nano-NPK in both seasons. For the combination treatments, the second treatment of vermicompost control plus 2 gL−1 NPK gave the maximum Fixed oil percentage of Moringa oleifera seeds (38.69 and 37.48%) with insignificant differences between this result and that of vermicompost control plus NPK control in the first and second seasons, respectively.The fixed oil percentage of  seasons . ContrarMoringa oleifera tree in response to treatment with vermicompost and NPK and their combination is presented in −1 vermicompost recorded the maximum mean values of fixed oil yield per tree (176.4 and 189.9 mL plant−1) and per hectare (1764 and 1899 L ha−1) in the first and second seasons, respectively, as compared with the other vermicompost levels and control. In the first season, there were insignificant differences between the applications of 50 and 60 ton ha−1 vermicompost in terms of fixed oil yield per tree and per hectare in the first season. NPK fertilization was able to increase fixed oil yield per tree and per hectare successfully in the first and second seasons as compared with the control treatment. Moreover, the foliar application of Nano-NPK produced the maximum mean values of fixed oil yield per tree (155.1 and 170.7 mL plant−1) and per hectare (1551 and 1707 L ha−1) in the first and second seasons, respectively. For the combination treatments, the application of 50 ton ha−1 vermicompost plus 2 gL−1 nano-NPK by spraying gave the maximum mean values of fixed oil yield per tree (270.5 and 299.1 mL plant−1) and per hectare (2705 and 2991 L ha−1) in the first and second seasons, respectively. Insignificant differences were detected among T15, T18, and T21 treatments in fixed oil yield per tree and per hectare in the first season of the study.The variability of seeds fixed oil yield per tree and per hectare of field grown the Moringa oleifera fixed oil are stearic acid, palmitic acid, eicosenoic acid, behenic acid and lignoceric acids (Moringa oleifera fixed oil increased by increasing the vermicompost level. The 10 ton ha−1 vermicompost treatment produced the minimum percentage of stearic acid (3.71%), palmitic acid (4.47%); eicosenoic acid (2.07%), behenic acid (4.40%), and lignoceric acid (0.64%). With NPK treatments, the control treatment had the minimum mean percentages of stearic acid (4.02%), palmitic acid (4.51%), eicosenoic acid (2.25%), behenic acid (4.63%) and lignoceric acid (0.68%). With the combination treatment, the minimum percentage of stearic acid (3.00%), palmitic acid (4.20%), eicosenoic acid (1.91%), behenic acid (3.81%) and lignoceric acid (0.14%) were recorded with the application of the 20 ton ha−1 vermicompost plus NPK control treatment.The major of detected saturated fatty acids of ic acids . In thisMoringa oleifera fixed oil are oleic acid, linoleic acid, α-linolenic acid, palmitoleic acid and paullinic acids , palmitoleic acid (2.55%), and paullinic acid (2.61%) were recorded with the 20 ton ha−1 vermicompost treatment. Using NPK, the control gave the maximum mean percentage of oleic acid (70.09%), linoleic acid (3.56%), α-linolenic acid (0.58%), palmitoleic acid (2.20%) and paullinic acid (2.45%). Regarding the combination treatments, the maximum highly significant mean percentages of oleic acid (72.46 and 72.56%) were found with the fourth and seventh combination treatments, respectively. In addition, the maximum mean percentages of linoleic acid (4.18%), α-linolenic acid (0.83%), palmitoleic acid (3.13%), and paullinic acid (2.78%) were recorded with the seventh combination treatment.The main unsaturated fatty acids of ic acids . By compMoringa oleifera tree were able to germinate under the conditions of calcareous soil without any organic or inorganic fertilization treatment ; however, after that the seedlings grew but very slowly. Finally, they produced small trees looking like thin branches with a few leaves with very small inflorescences. This gave the minimum mean values of pod and seed parameters, and yield of mature pods, seeds, and fixed oil per tree and per hectare in both seasons. This may have been due to the lack of accessibility of soil nutrients resources in the calcareous soil where nitrogen had been lost via leaching, deep percolation, or N transformations. Moreover, there was low availability of phosphorous and micronutrients have low availability and there was an imbalance among potassium, magnesium, calcium, and other elements [−1 vermicompost plus 2 gL−1 NPK, with insignificant differences between them, since the control treatment gave very small semi-atrophied seeds. As mentioned before, in calcareous soil, the amount of nitrogen and its availability are limited [In this experiment, the seeds of the elements ,11,53, aelements . These relements , who fouelements  reportedelements  found thelements  found th limited ,11,54. U limited , and decMoringa oleifera tree, with its low rate of nutrients degradation speed [It was obvious from the obtained results that the improvements of all the studied parameters of pods and seeds and the yield of pods, seeds, and fixed oil per tree and per hectare increased when the amount of vermicompost used was increased in the two seasons of the study. These improvements may have been due to the role of vermicompost in regulating growth by its natural auxins, gibberellins, and cytokinins contents, and increasing the availability of plant nutrients such as nitrogen, phosphorus, potassium and micro-elements in the soil through the mineralization of organic matter; increasing soluble forms of nutrients by improving the soil pH; as well as increasing the uptake of elements by roots ,58,59. Ion speed ,60. The on speed ,65,66,67on speed , Aranconon speed , Atiyeh on speed , Liuc anon speed  and Muscon speed  who founEven though increased vermicompost improved the yield of fixed oil as a result of improving plant growth, it decreased the percentage of fixed oil of seeds, which may be because under high N conditions, the metabolism of grown plants shifts towards production of N-compounds, with reduced production of secondary non-N-containing metabolites such as phenolics, fatty acids, and terpenoids ,73. Wher2 assimilation rate in plants through its role in opening and closing stomata. Thus, it enhances photosynthesis and controls in the amount of glucose produced in plants and its translocation to seeds by controlling the enzymes of carbohydrate metabolism [Moringa oleifera tree as compared with the other ground application treatments and the control in both seasons under alkaline calcareous soil conditions. On the one hand, foliar fertilization has better potential to correct nutritional deficiencies in plants caused by the improper supply of nutrients to roots, and this practice is usually more economical and effective under alkaline calcareous soil conditions [Moringa trees encouraged root development and leaf canopy growth. Liu and Lal [Glycine max. Fagbenro [An inadequate supply of nitrogen, phosphorus and potassium during crop growth is known to have a negative impact on the reproductive capability, growth, and yield of plants ,78,79. Ttabolism ,84,85. Tnditions ,87,88. Onditions . Nanofernditions ,91, and nditions . Sprayinnditions ,93,94. Tnditions  and Dixonditions  suggeste and Lal  reportedFagbenro , Ainika Fagbenro , GhafariFagbenro , MahmoodFagbenro , and DelFagbenro  reportedFagbenro , Farnia Fagbenro , OyedejiFagbenro , BărăscuFagbenro , and MokFagbenro  comparedFagbenro  recordedFagbenro  found thFagbenro  reportedFagbenro , SoleimaFagbenro , Arif etFagbenro , and HamFagbenro  reportedFagbenro  found thFagbenro  recordedFagbenro  reportedMoringa oleifera trees. Despite the important role of foliar NPK application in terms of rapid assimilation and translocation and the positive influence on growth and yield, foliar fertilization cannot replace nutrition through the roots;. however, it can be used to reduce the use of fertilizers on the soil . To over−1 NPK gave the maximum fixed oil percentage. In addition, the minimum mean percentages of stearic acid, palmitic acid, eicosenoic acid, behenic acid and lignoceric saturated fatty acids and the maximum mean percentages of oleic acid, linoleic acid, α-linolenic acid, palmitoleic acid, and paullinic unsaturated fatty acids were recorded with the application of the combination treatment of 20 ton ha−1 vermicompost plus the NPK control. The combination of organic and inorganic fertilizers, generally, has vital effects on plant growth as well as soil chemical and biological properties [In this study, combination treatments of vermicompost plus NPK and nano-NPK improved pod and seed parameters and yield per tree and per hectare, and these improvements increased with an increased vermicompost level and with spraying nano-NPK, while treatment of vermicompost control plus 2 gLoperties ,118 geneoperties .Moringa oleifera seeds was up to 40% with a high-quality fatty acid composition, as the percentage of unsaturated oleic fatty acid reached 70% or more. In this study, the percentage of oleic acid decreased with increased fertilization level. These results were in agreement with those of Xie et al. [Our results were in agreement with those of Bajracharya et al. , Bhattare et al.  on flax e et al.  on blackMoringa oleifera trees. The present investigation was carried out during two successive seasons in 2018/2019 and 2019/2020 in an open field of a private farm in El-Amiriya, Alexandria Governorate, Egypt. The aim wasto study the effects of organic and inorganic fertilization on pod, seed and fixed oil yield as well as composition, especially the percentage of oleic fatty acid of the Moringa oleifera tree grown alone in an isolated place in the study location. Its seed was previously brought from the national research center. The seeds were cultivated in February 2018 and 2019. A drip irrigation system was applied. Soil drainage conditions at the site were adequate to guarantee good oxygenation of the crop.Seeds were collected from one selected mature Moringa oleifera plants were assigned to organic fertilization in the form of a ground dose of vermicompost and sub-plots were assigned to mineral fertilization in the forms of mineral and nano-NPK (19:19:19). All possible combinations of the two studied factors were made  and NPK fertilization (0 (control) and 2 gL−1 NPK or 2 gL−1 Nano-NPK). Every studied amount of vermicompost was added before planting over ten days, while the 2 gL−1 NPK treatment was applied as a ground dose and 2 gL−1 Nano-NPK was applied as foliar application. All NPK treatments were applied once per week after two weeks and until six weeks from planting; after that, they were applied twice a week until the end of the experiment. Tween 80 (0.01%) was used as the wetting agent. Untreated plants (NPK control) and plants treated with 2 gL−1 NPK were sprayed with distilled water and Tween 80 (0.01%). The data are presented as mean values ± SE (n = 3). The experiments were conducted in a split plot arranged in a Randomized Complete Block Design (RCBD) with three replications during February and March 2018/2019 and 2019/2020. The main plots of the ere made . The expst February in both seasons. Around 400 g of 19:19:19 NPK mineral fertilizer was weighed in a 2 L glass beaker, then 550 mL of distilled water was added, and it was stirred until completely dissolved. The clear solution was heated to 50 °C, and with vigorous stirring, 50 g of citric acid was added and stirring was continued for 15 min. Potassium hydroxide was added slowly until the desired pH was reached. During the addition of potassium hydroxide, the clear solution changed to a milky appearance, indicating the conversion to nanoparticle size. The concentration used was prepared according to the amount of mineral NPK used in the preparation of nano-NPK. Seeds were sown on 1−1) were sown on one side of the row. After 30 days, the seedlings were thinned to one plant per hill. The plots were weeded every two weeks when possible. The climate of the culture location is desert, with a mean annual temperature of 20.8 °C and annual precipitation of 181 mm, mainly falling in November through February [n = 3).Seeds , number of seeds per pod, weight of mature seed weight, and yield of mature seeds (g tree−1 and kg ha−1). The data are presented as mean values ± SE (n = 3). A sample of five plants was taken at random from each replication and fifteen plants from every treatment to measure the following parameters: number of pods per inflorescence, number of pods per tree, weight of mature pods (g), yield of mature pods (g treen = 3). To determine the fixed oil content, seeds of each treatment were randomly selected, weighed, and dried at 50 °C. The drying process was continued until the difference between the two successive weights was less than 1 mg. Three replications were used for this process. The oil was extracted over 16 h with hexane using a Soxhlet apparatus . The perFatty acid methyl esters were prepared with methanolic sulfuric acid and characterized by gas chromatography mass spectrometry. The analyses of the fixed oil were conducted using a gas chromatography–mass spectrometry (GC-MS) instrument at the Department of Medicinal and Aromatic Plants Research, National Research Center as mentioned in Atteya and Amer The experiments were a split plot arranged in a Randomized Complete Block Design (RCBD) with three replicates. Analysis of variance with SAS software  was carrMoringa oleifera pods and seeds. Finally, in this study, the recommended treatment for reaching the maximum values for the yield of mature pods, seeds, and fixed oil per tree and per hectare is the 50 ton ha−1 vermicompost plus 2 g L−1 Nano-NPK treatment. The 20 ton ha−1 vermicompost plus NPK control treatment is recommended for producing fixed oil with the minimum percentage of saturated fatty acids and the maximum percentage of oleic acid.As Compared with the soil-application of NPK, in this study, the foliar application of nano-NPK provided a good resolution for a low availability of NPK. Moreover, increasing the amount of vermicompost improved the parameters and yield of


Anal sphincter incontinence is a chronic disease, which dramatically impairs quality of life and induces high costs for the society. Surgery, considered as the best curative option, shows a disappointing success rate. Stem/progenitor cell therapy is pledging, for anal sphincter incontinence, a substitute to surgery with higher efficacy. However, the published literature is disparate. Our aim was to perform a review on the development of cell therapy for anal sphincter incontinence with critical analyses of its pitfalls. Animal models for anal sphincter incontinence were varied and tried to reproduce distinct clinical situations  but were limited by anatomical considerations. Cell preparations used for treatment, originated, in order of frequency, from skeletal muscle, bone marrow or fat tissue. The characterization of these preparations was often incomplete and stemness not always addressed. Despite a lack of understanding of sphincter healing processes and the exact mechanism of action of cell preparations, this treatment was evaluated in 83 incontinent patients, reporting encouraging results. However, further development is necessary to establish the correct indications, to determine the most-suited cell type, to standardize the cell preparation method and to validate the route and number of cell delivery. Anal sphincter incontinence (ASI) is defined by an involuntary loss of gases, solids and/or liquid materials. The prevalence of ASI is widely underestimated and ranges from 0.4 to 24% . ASI is The physiopathology of ASI is not completely understood but results from a functional impairment of one or more of the four anatomic components that allow a normal continence: The internal anal sphincter (IAS), the external anal sphincter (EAS), the pelvic floor muscles and the sensory-motor apparatus . ASI is In patients with ASI, there are at least two mechanisms leading to a modification in the pelvic floor structure or function. First, sphincters can be injured by perineal tearing, stretching and/or ischemia; obstetric trauma being the most common cause . The epiThe classical clinical situation of acute anal sphincter injury is well illustrated by childbirth trauma, which still occurs in 11% of vaginal deliveries and can extend up to the IAS and sometimes the rectum   Figure . In the Progenitor cells produce cytokines like stromal-derived factor 1 (SDF-1) that are both chemoattractive for progenitor cells (including myoblasts and smooth muscle progenitor cells) but also contribute to cell proliferation, migration and survival ,57,58. SIn theory, progenitor cells could be transplanted soon after injury and this is considered to be the best option to maximize the effect of transplantation  20]. In. In20]. Most often, an anal sphincter tear is usually identified after delivery and surgically repaired . SometimAnal sphincters encircle the anal canal, and the loss of this circular shape, as a result of injury, directly impairs the continence function . HoweverIn the majority of preclinical studies, researchers investigated the direct injection of mesenchymal stem cells (MSC) in the area of muscle injury . This apAnother strategy to treat patients with ASI is the transplantation of a biosphincter. Bitar’s group, from the Wake Forest Institute for Regenerative Medicine, has been working for decades on the implantation of bioengineered anal sphincters composed of innervated smooth muscle cells . The resPatients with visible EAS or IAS sphincter lesions are good candidates for surgical repair. Park’s sphincteroplasty is the most common procedure where the two edges of the damaged sphincters are brought together with an overlapping suture ,63. UsuaIn some patients with ASI, a clear injury event cannot be found, and patients have either weak anal sphincter tonus without an anomaly on imaging or hypotrophy of anal sphincters on imaging. This could be the result of aging, denervation or degenerative mechanisms inducing a chronic injury to the anal sphincters . IAS is Diabetes has been associated with ASI  and may To date, there is unfortunately no model mimicking a degeneration of the muscles of EAS or IAS. Hosokawa et al. proposed a model of EAS and IAS injury using cardiotoxin but the injury was finally similar to sphincter resection . This moMultipotent cells proposed for ASI cell therapy can be derived from various tissues. The most frequent sites were skeletal muscle, bone marrow or adipose tissue. Cell preparations were either syngeneic or autologous, and allogeneic or xenogeneic transplantation was marginal . Cells from a muscular origin were used in the majority of the identified publications: In 17 studies, they originated from skeletal muscle or EAS ,91,92,93Cells of bone marrow origin were used in 10 studies ,43,46,48+ interstitial cells and fibro-adipogenic progenitors . T. T104]. genitors .Satellite cells have the ability to reform muscle fiber  and haveCells derived from adipose tissue constitute an interesting option, as subcutaneous fat tissue is easily accessible. The cells used in the reported studies ,34,35,71Stem cell and progenitor cells were retrieved from rats, mice, rabbits, non-human primate or humans . DiffereThe characterization of isolated multipotent cells is mandatory for reproducibility but also for quality purpose, especially when a clinical application for ASI treatment is foreseen. Among publications using muscle multipotent cells, only 13 studies reported or referred to a proper characterization of the cells and were heterogeneous for markers  36,37,4,68,69,93+ and CD45− [Cells of bone marrow origin were used in nine publications ,43,46,48nd CD45− ,35,38,71nd CD45−  but thernd CD45− . Thus, mBefore transplantation, cells were cultivated on plastic dishes and the number of passages before injection in the identified clinical trials ranged from three to ten. The number of injected cells, clearly reported by identified in vivo reports, ranged from 10,000 up to 90 million. In clinical trials, this number ranged from 200,000 up to 2 billion. However, the minimum number of cells required to obtain a beneficial effect on ASI remains elusive and only a few authors performed a real titration ,92. ThusAs a strategy to limit cellular stress due to transplantation, some authors proposed to protect cells with biomaterials ,54,110. As mentioned earlier, in some studies, authors performed immediate injection of cells along with sphincter repair, confronting cells with an acute inflammatory environment that presumably precludes cell survival . On the Further, growth factors  described to promote stem cell implantation were delivered in situ by bioscaffolds or through osmotic pumps ,100,103.For in vitro studies, physiological functional evaluation was carried out in almost all identified studies to assess the contractility potential of constructed sphincters. For in vivo studies, outcomes were highly variable: The most common methods for outcome assessment were histology, anorectal manometry, physiological functionality evaluation and electromyography or electrophysiology. Some authors tracked cells using magnetic resonance imaging  or label® device, Medtronic, Dublin, Ireland [The determination of the outcome in a clinical study on anal incontinence is challenging. Different definitions of ASI exists, including incontinence to gases or not . Further Ireland ).A review of the literature identified a total of 52 original publications. Seven publications reported in vitro results  79,93,9,95,97,98p > 0.001). In addition, anal manometry demonstrated an improvement of median resting and squeeze pressures (20 (IQR 17–28) to 32 (25–43) and 23 (IQR 20–34) to 33 (IQR 31–66), respectively). The same group started a second trial including 34 females and 5 males and found a reduction of Wexner score of −16.2 (SD-3.66) for women and −18.8 (SD-1.30) for male at one year. These results were better than sphincteroplasty, which typically induces a long-term reduction of −1 to −5.2 of median/mean Wexner score (mean follow-up between seven and eight years) [p = 0.006) for the intervention group and a reduction of −1 (range −8 to 6) (p = 0.35) for the placebo [Almost all patients included in clinical trials exhibited EAS injury. Four out of seven studies used the Wexner score as the primary outcome. The FIQL score was used in two studies. Other variables measured were anorectal manometry (5/7), endoanal ultrasonography (3/7) and electromyography (EMG) (3/7). The longest follow-up was reported by the group of Frudinger et al. who injected autologous myoblasts into EAS in 10 voluntary women with EAS defect or atrophy . After at years) ,125. In  placebo . However placebo . GloballUntil now, in vivo experiments have demonstrated a relative oncological safety of such a strategy. One in vivo study described focal cell growth at the injection site but without malignant characteristics . Recent Preclinical and clinical studies have demonstrated the safety of multipotent cell utilization for ASI. Published clinical studies have shown promising results, but only three of them were controlled with placebo injection ,71,72. TThe ideal therapy for ASI should be cost-effective with a long-lasting effect. Apart from research and development costs, good manufacturing practices, GMP certifications and implementation charges, routine use of cell therapy appears to be highly costly . Trébol After transplantation, progenitor cells and stem cells might act by paracrine effects and/or by differentiation into functional muscular cells. It should be pointed out that the exact underlying mechanism remains poorly understood, and that basic research on this topic is still required to understand which factors and conditions are leading to cell engraftment, differentiation and finally tissue regeneration ,131.Moreover, the natural history of sphincter lesion/repaired sphincter healing should be better understood in order to select appropriate cell preparations and transplantation techniques. Some groups reported a different approach, considering the use of cell therapy as an add-on to sphincter repair, either directly after lesion, to simulate the primary repair of an acute obstetrical tear or at distance ,31,32,33


Derived from magnetotactic bacteria (MTB), magnetosomes consist of magnetite crystals enclosed within a lipid bilayer membrane and are known to possess advantages over artificially synthesized nanoparticles because of the narrow size distribution, uniform morphology, high purity and crystallinity, single magnetic domain, good biocompatibility, and easy surface modification. These unique properties have increasingly attracted researchers to apply bacterial magnetosomes (BMs) in the fields of biology and medicine as MRI imaging contrast agents. Due to the concern of biosafety, a long-term follow-up of the distribution and clearance of BMs after entering the body is necessary. In this study, we tracked changes of BMs in major organs of mice up to 135 days after intravenous injection using a combination of several techniques. We not only confirmed the liver as the well-known targeted organs of BMs, but also found that BMs accumulated in the spleen. Besides, two major elimination paths, as well as the approximate length of time for BMs to be cleared from the mice, were revealed. Together, the results not only confirm that BMs have high biocompatibility, but also provide a long-term in-vivo assessment which may further help to forward the clinical applications of BMs as an MRI contrast agent. Themembrane ,6,7,8. Tmembrane ,10. BMs membrane . The promembrane ,12,13. Iondition . Studies on the applications of BMs are increasing in recent years, especially in the biomedical field. As superparamagnetic nanoparticles, BMs were reported to have higher transverse relaxivity (r2) and more effective signal decay than chemically synthesized nanoparticles as contrast agents for magnetic resonance imaging (MRI) application ,16,17. SAs an MRI contrast agent, applications of BMs in vivo require systematic studies on the toxicity and biocompatibility, including their distributions and clearance after intravenous injection. However, so far, most researches focus on the potential applications of BMs, and few have reported their toxicity and biosafety. Sun et al. examined for the first time the systematic effect of BMs on rats for the acute toxicity, immunotoxicity, and cytotoxicity of BMs from MSR-1 . HoweverIn this study, we have performed long-term in-vivo and in-vitro experiments to track the distribution and clearance of BMs. We monitored BMs after intravenous injection for 135 days by magnetic resonance imaging (MRI) of mice and found that BMs could cause fast signal decay in T2-weighted images of the liver and spleen, and the signal was gradually recovered with the clearance of BMs, indicating that the targeted organs were liver and spleen. Prussian blue staining and Haematoxylin and Eosin (HE) staining of tissues further verified MRI results and further showed good biocompatibility of BMs. Moreover, inductively coupled plasma mass spectrometry (ICP-MS) was applied to quantitatively analyze the iron content at different time points after intravenous injection. Together with the degradation of BMs in cells observed by immunocytofluorescence (ICF) imaging, a picture of the distribution and elimination paths of BMs in mice was obtained. We expect that this work will help to understand the biocompatibility and in-vivo behavior of BMs, and can provide a reference for the future clinical applications of BMs.Magnetospirillum gryphisiwaldense MSR-1  was obtained from China Agricultural University. The wild-type magnetotactic bacteria MSR-1 were cultured at 30 °C and 100 rpm/min in the medium as described in our previous study [us study . The bacus study ,31,32. ATwo healthy New Zealand rabbits were used to prepare polyclonal antibodies against the surface proteins of BMs, which were then used for the localization of BMs in subsequent Immunocytofluorescence (ICF) assay. The rabbits were immunized with 1 mL BMs (2 mg/mL) that were fully emulsified with the same dose of Freund’s complete adjuvant and injected into the back of rabbits at multiple points for the first immunization. Then 1 mL BMs (0.5 mg/mL) were fully emulsified with the same dose of incomplete Freund’s adjuvant to enhance the immunization every 2 to 3 weeks. Afterwards, the serum of rabbits was taken for Elisa detection after three immunizations. If the antibody titer was above 1:16,000, all serum would be collected for purification. If the antibody titer was lower than 1:16,000, the immunization strengthening step was continued until qualified. Finally, all serum of each rabbit was taken and purified by protein affinity column chromatography.TEM  was used to visualize the morphological characteristic of BMs. BMs were washed three times with ultrapure water and resuspended in ultrapure water to make a stock solution, then diluted to an appropriate concentration with ultrapure water. The sample was dropped on a duplex copper grid coated with carbon film to incubate for 3 min and air-dried at room temperature. Images were recorded at an accelerating voltage of 80 kV.Zetasizer Nano ZS  was used to determine the surface charge and the size distribution of BMs dispersed in ultrapure water. All measurements were conducted at room temperature.2 atmosphere. When cells reached 80% confluence, 30 μg/mL of BMs were added and incubated for 24 h before conducting the follow-up experiments.Raw 264.7 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium , 10% fetal bovine serum , and 1% penicillin/streptomycin  at 37 °C in a humidified 5% COTo visualize the intracellular localization of BMs, sulfo-cyanine5 NHS ester  was used to label BMs. Raw 264.7 cells were incubated with labeled BMs at 37 °C for 3 h. After incubation, Raw 264.7 cell membrane was stained with DIO membrane fluorochrome at 37 °C for 20 min , and the cell nucleus was stained with Hochest 33342  at 37 °C for 10 min, and then observed the images by laser confocal microscope .To check the fate of BMs after entering the cell, Raw 264.7 cells were seeded in confocal dishes at 80% confluence and incubated with BMs (30 μg/mL) for the designated time. The cells were collected and confocal laser imaging was performed at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, 144 h, respectively. Specifically, the supernatant of cells was discarded and replaced with fresh medium at 6 h to remove extracellular BMs. at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, 144 h, respectively (the supernatant of cells was discarded and replaced with fresh medium at 6 h). After incubation, cells were washed with PBS  and fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% Triton X-100 for 20 min and blocked with 10% bovine serum albumin (BSA) for 1 h at room temperature. Afterwards, cells were incubated with the anti-magnetosomes antibody overnight at 4 °C, then washed with PBS three times, and incubated with FITC goat anti-rabbit antibody  at room temperature for 1 h. Finally, cells were stained with DAPI  for 5 min and images were obtained by laser confocal microscope .Raw 264.7 cells incubated with BMs were subjected to Prussian blue staining  to evaluate the degradation of the inorganic core of BMs inside the cells. The cells were collected and Prussian blue staining were performed at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, 144 h, respectively (The culture medium was discarded and replaced with the fresh one at 6 h). Cells were first fixed with 4% paraformaldehyde for 15 min and stained with Prussian blue dye by incubating at 37 °C for 30 min, followed by washing with ultrapure water twice and stained with Prussian blue counterstain for 30 to 60 s. Finally, cells were washed with ultrapure water and imaged by EVOS microscope . All animal experiments were conducted in accordance with the institutional guidelines approved by the Institutional Ethical Committee of Animal Experimentation of the National Center for Nanoscience and Technology. Animals received care following the Guidance Suggestions for the Care and Use of Laboratory Animals. Healthy male 6–8week-old C57BL/6NCrl (C57) mice, SPF grade, were provided by Charles River Laboratories. Laboratory animal license number: Beijing SYXK 2017-0033 Beijing SYXK 2017-0022.2, ETL: 10, and MTX: 256 × 256.The 6–8 week-old male C57 mice were divided into three groups with three mice in each group for MRI. The clinical dosage of ferroferric oxide is around 7.5 μmol Fe/kg . Sun et 2O2 for 25 min). Afterwards, the sample was incubated with F4/80 primary antibody, goat anti-mouse secondary antibody labeled with HRP, and finally stained with DAB. HE staining was performed following a standard protocol. The staining results were captured by microscope .Haematoxylin and eosin (HE) staining and immunohistochemical (IHC) analysis were performed on paraffin-embedded sections of mice liver and spleen tissues. Tissues were first fixed by immersion in 4% paraformaldehyde, then embedded in paraffin. And Paraffin sections were dewaxed with xylene, gradient alcohol hydration. After dewaxing and hydration, endogenous peroxidase blocking was carried out . At the designated time point, three mice were sacrificed, and the liver, spleen, heart, lung, kidney, intestine, and brain from each were removed, fixed with paraformaldehyde, and embedded with paraffin for ultra-thin sectioning. The ultra-thin sections were stained with Prussian blue and images were acquired.The inductively coupled plasma-mass spectrometry (ICP-MS) analysis was performed with a Thermo ICAP-QC Series ICP-MS. The 6–8 week-old male C57 mice were divided into ten groups with three mice in each group for detecting Fe concentration. The mice were injected intravenously with magnetosome suspension at a dosage of 32 mg/kg (20 mg Fe/kg). Then, the liver, spleen, blood, urine, and feces samples were obtained at the designated time point. To prevent the blood remaining in the tissues from interfering with the determination of iron level, cardiac perfusion was performed before harvesting tissues. Mice were anesthetized by injecting 200 μL 4% chloral hydrate into the abdominal cavity. The limbs of mice were fixed on the anatomical table. The xiphoid process was then lifted with tweezers, the chest was cut with scissors, and the pericardium was torn open with tweezers, exposing the heart. The right auricle was cut open with scissors, and the prepared saline syringe was inserted into the left ventricle to conduct the perfusion. The perfusion was ended when the fluid out of the right auricle was clarified.p values of less than 0.05 were considered to be statistically significant. All the quantitative data in our studies were subjected to statistical analysis and were expressed as mean ± standard deviation (SD). The statistical analysis was performed with GraphPad Prism statistics software. Magnetospirillum gryphisiwaldense MSR-1 magnetotactic bacteria by the ultrasonic crushing method [The BMs were purified from g method . The traTo track the distribution and clearance of BMs after tail administration, magnetic resonance imaging (MRI) was performed on mice at the designated time points from day 0 to day 62 and 135 for the low and high dose, respectively, when the transverse relaxation time (T2) nearly returned to the control level . The resTo confirm BMs targeted organs and cells after being injected into the mice, we conducted Prussian blue staining on mice tissues after the continuous ultra-thin section. Briefly, at the designated time point, mice were sacrificed, and the heart, liver, spleen, lung, kidney, brain, and intestine were obtained for the ultrathin sectioning. Additionally, macrophages in the liver and spleen were subjected to immunohistochemical staining using F4/80 as the marker. The Prussian blue results showed that BMs were mainly accumulated in the liver and spleen after entering and circulating in the blood. No blue coloration was founded in other organs, indicating few BMs were accumulated at other parts of the body . BMs werNext, hematoxylin-eosin (HE) staining was carried out to determine whether the BMs cause damage to the liver and spleen after tail vein injection into mice. No infiltration of inflammatory cells, deposition of foam cells, plaques, cell degeneration, and necrosis were founded E, indicaQuantitative measurement of the iron element using inductively coupled plasma mass spectrometry (ICP-MS) was performed to detect changes of the iron level in the liver, spleen, blood, urine, and feces after injection of BMs at the dose of 32 mg/kg. Since the blood has a high level of iron, to reduce the interference of the blood in organs, cardiac perfusion was carried out to flush out the blood in the organs until the color of the liver changed from purple to khaki.The results showed that the iron level in the liver increased sharply in a short time after intravenous injection of BMs, from 10–20 mg/kg to more than 400 mg/kg. The iron level in the liver was the highest in the first two days and began to decrease, but remained significantly high for 30 days, and returned to the base level gradually A. In theThe colocalization of BMs with macrophages in the liver and spleen suggested they entered macrophages, resulting in the accumulation in the liver, spleen, and blood that have a large number of macrophages. In the meantime, the iron level peak in the urine should be contributed by iron ions, suggesting BMs undergo degradation in the macrophages.To verify that BMs were internalized by macrophages, we performed the immunofluorescence (IF) assay to observe the localization of labeled BMs. As shown in To analyze the degradation of magnetosomes in cells, we conducted immunocytofluorescence (ICF) experiments using antibodies against BMs to detect the membrane integrity of BMs at different time intervals after incubation of BMs with Raw 264.7. The inorganic iron core was also monitored using Prussian blue staining. Laser confocal imaging of ICF showed that green fluorescence signals enhanced apparently at 3 h after incubation with BMs, the intensity increased as the incubation continued and lasted until 12 h . The sigTo check the degradation of BMs inorganic core in cells, we performed the cellular Prussian blue staining. As shown in In this study, we tracked the distribution and clearance of bacterial (MSR-1) magnetosomes in major organs for up to 135 days after intravenous injection into mice using MRI and various methods. MRI signal decay can be detected in the first two days for the low dosage and a few weeks for the high dosage. This is expected because almost half of BMs went to feces in the first two days (discussed below) and the particles uptaken by macrophages were mostly eliminated in 72 h. The consistency of MRI results and others confirms the high sensitivity of BMs as a contrast agent. ICP-MS measures both BMs and iron ions and is very sensitive; besides systemic measurement errors, the fact that BMs are not evenly distributed in the liver and spleen, variations of the individual mouse, and the slight difference of the excretion amount have caused variations of the quantitative ICP-MS results. Therefore, the averaged results only reflect trends of changes in different tissues and organs over time, which are consistent with MRI and Prussian blue staining results.The clinical dosage of ferroferric oxide as an MRI contrast agent is approximately 7.5 μmol Fe/kg body weight . Since BOur results are consistent with Sun et al. who reported BMs accumulated in the liver, but they did not find BMs present in other organs . HoweverThe clearance time of BMs in mice was positively correlated with the injection amount. When the amount was 8 mg/kg, the majority of BMs were eliminated from the liver at around 30 days after injection. However, at the dosage of 32 mg/kg, the clearance took 120 days. From the ICP-MS, we estimated that approximately 1/2 of the injection infiltrated the bile duct and went to the feces in the first two days, while the remaining BMs were degraded into smaller particles and eventually became iron ions and eliminated through urination . The cleTo summarize, we reported for the first time a long-term follow-up of the distribution and clearance of BMs in different tissues and organs in mice using a combination of several methods including MRI. Our study further confirmed that BMs as an MRI contrast agent have sufficient spatial resolution and high sensitivity. Meanwhile, we found the major targeting organs of BMs, the length of time, and two main paths for BMs to be cleared from mice. The targeting and biodegradability of the BMs suggest that it has the potential as a contrast agent for imaging liver and spleen, or other organs and tissues for disease treatment or diagnosis after modification to avoid macrophage uptake. The results may be useful in the clinical application and contribute to the biosafety assessment of BMs.