Butyrate, a by-product of gut bacteria fermentation as well as the digestion of fat in mother’s milk, exerts a wide spectrum of beneficial effects in the gastrointestinal tissues. The present study aimed to determine the effects of sodium butyrate on small intestine contractility in neonatal piglets. Piglets were fed milk formula alone (group C) or milk formula supplemented with sodium butyrate (group B). After a 7-day treatment period, isometric recordings of whole-thickness segments of the duodenum and middle jejunum were obtained by electric field stimulation under the influence of increasing doses of Ach (acetylocholine) in the presence of TTX (tetrodotoxin) and atropine. Moreover, structural properties of the intestinal wall were assessed, together with the expression of cholinergic and muscarinic receptors (M1 and M2). In both intestinal segments (duodenum and middle jejunum), EFS (electric field stimulation) impulses resulted in increased contractility and amplitude of contractions in group B compared to group C. Additionally, exposure to dietary butyrate led to a significant increase in tunica muscularis thickness in the duodenum, while mitotic and apoptotic indices were increased in the middle jejunum. The expression of M1 and M2 receptors in the middle jejunum was significantly higher after butyrate treatment. The results indicate increased cholinergic signaling and small intestinal growth and renewal in response to feeding with milk formula enriched with sodium butyrate in neonatal piglets. In mammals, the gastrointestinal tract (GIT) is both structurally and functionally immature at birth; therefore, its development continues in the early postnatal period. During this time, the intestine undergoes adaptive processes associated with significant changes in structural growth and remodeling of the intestinal epithelium as well as functional maturation, including changes in intestinal absorption and motor activity. It should also be mentioned that approximately one-third of preterm and low birth weight infants display delayed maturation of motility patterns within the gut .Breastfeeding is the best nutritional strategy for infants. In contrast to milk formula, mother’s milk provides the newborn with biologically active compounds, which optimize gut microbial colonization, support immune maturation and gastrointestinal growth and development, program metabolism, and influence cognitive development. Moreover, feeding with mother’s milk prevents both short-term and long-term health complications, including necrotizing enterocolitis in preterm neonates as well as obesity, diabetes, and cardiovascular diseases later in adult life. However, there is still a relatively large group of infants who require milk formula feeding, and thus efforts should be made to improve its nutritional quality .Butyrate is a four-carbon, short-chain fatty acid (SCFA) that originates from gut bacterial fermentation. During the suckling period, the majority of butyrate is formed as a by-product from the gastric digestion of fat in the mother’s milk . SimilarButyrate exerts a number of beneficial effects on the intestinal epithelium that extend far beyond the colon. Butyrate serves as a fuel source for epithelial cells, exerts trophic effects on the intestinal mucosa, reduces gut inflammation, strengthens the gut barrier function, stimulates secretion of pancreatic and jejunal enzymes, modifies ileal microbiota, and facilitates apoptosis of colonic cancer cells ,11,12. MThese data highlight the important role of butyrate in the maintenance of intestinal health and at the same time indicate the lack of evidence on how butyrate affects GI motility in neonates. Thus, the aim of this study was to determine the effects of sodium butyrate on the motor activity of duodenum and jejunum in newborn piglets fed milk formula.The study was approved by the University of Lund Ethics Review Committee on Animal Experiments . Animal husbandry and care were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.The experiment used 16 newborn male piglets (Yorkshire × Swedish Landrace) × Hampshire) obtained from a SPF local herd . They were born on time and without complications, with an average body weight (BW) of 1.95 ± 0.22 kg. For the first 24 h of life, the piglets were kept with their sows and then transported to the Lund University animal facility and housed in individual cages. The cages were equipped with an artificial sow system , which provided each piglet with fresh milk formula  every 75 min (20 times/day). The amount of milk formula administered was calculated based on daily BW gain and protein requirements of newborn piglets (11.0–11.3 g/kg).n = 8): control group (C), which was fed milk formula alone, and the experimental group (B), which was fed milk formula and supplemented with sodium butyrate  at a dose of 0.3 g/100 g dry matter. Piglets’ BW was recorded daily. At the end of a 7-day treatment period, piglets were euthanized by an overdose of pentobarbiturate . The GIT tissues were removed and collected for later analysis.After an adjustment period of 24 h, piglets were randomly allocated to two groups , and placed on silane-treated glass slides. For immunohistochemistry and TUNEL assay, antigen retrieval was performed by microwave-heated incubation  in citrate buffer (pH 6.0).2, 5% CO2) [−5 M acetylocholine  for 1 min. Next, the tissues were washed and allowed to equilibrate and then treated with cumulative doses of Ach (10−9–10−4 M) for dose–response contractility recordings (−5 M), .Duodenum and middle jejunum sections (15 mm long) were collected and immediately placed in ice-cold Krebs–Henseleit solution. The intestinal segments were mounted in organ baths (25 mL)  filled with Krebs–Henseleit solution, maintained at 37 °C, and aerated with carbogen (95% O 5% CO2) . The intcordings . Half of−5 M) . At the end of each experiment, the samples were treated with 10−5 M ACh to check the viability of the tissue and then with 10−4 M isoproterenol to control tissue relaxation.Neuronal-mediated contractile effect was evoked through electric field stimulation (EFS). Following a 1 h equilibration period, tissues underwent EFS  for 10 s at 90 V with different frequencies , with 1 min intervals between each pulse . Half ofDuodenum and middle jejunum sections were fixed as described above, immersed in xylene and descending grades of alcohol, and routinely stained with H&E. Three slides were randomly selected for each intestinal section, and 30 measurements of the muscularis layer thickness were performed using a light microscope coupled with a digital camera and image analysis software .Intestinal epithelium apoptosis was assessed in paraffin-embedded middle jejunum segments using the TUNEL method as was described in detail in the manufacturer’s instructions . Apoptotic cells were identified and counted in at least 15 high-power fields using a confocal microscope . The apoptotic index was assessed as the ratio of apoptotic cells to the total number of epithelial cells of villi or crypts, respectively. The nuclei of cells were stained with DAPI.Middle jejunum segments were incubated with primary polyclonal rabbit anti-Ki67 antibodies  for 30 min at room temperature in a humid chamber . After wImmunostaining of cholinergic muscarinic receptors 1, 2, and 3  was performed in the middle jejunum segments frozen immediately after collection at −80 °C . Prior tp < 0.05. Mann–Whitney test or unpaired t-test was used to indicate statistical differences between groups. All analyses were performed using GraphPad Prism version 7.0a .The data were expressed as mean ± standard deviation (SD) with significance defined as p = 0.0031). The final BW of piglets was 2.50 ± 0.38 and 3.10 ± 0.32 kg in group C and B, respectively (p = 0.001). The supplementation of milk formula with sodium butyrate had no effect on milk intake. The results of the thickness of the muscularis mucosa layer have been show on the The initial BW of piglets was 1.92 ± 0.25 and 1.99 ± 0.18 kg in group C and B, respectively. Within the 7-day treatment period, a significant increase in BW of the piglets was noted in both groups, with an average daily gain of 81 ± 59 and 159 ± 55 g in group C and B, respectively  caused a dose-dependent increase in contractile amplitude of whole-thickness intestinal samples of duodenum and middle jejunum in both groups of piglets . The contractile response to cumulative doses of ACh was significantly higher in the butyrate-treated group compared to the control group in both the duodenum and middle jejunum. Moreover, pretreatment with atropine led to a significant decrease in the responsiveness in both intestinal segments from both experimental groups. The responsiveness after blockage with atropine was also significantly higher in the duodenum of group B compared to that of the control piglets  in the homogenates from the middle jejunum of newborn piglets .The intestine plays a key role in neonatal growth and health, including metabolic programming. In the present study, we showed that sodium butyrate stimulates small intestinal contractility in neonatal piglets fed milk formula lacking butyrate. To our knowledge, ours is the first study to demonstrate duodenal and jejunum contractility in newborn piglets in response to oral administration of sodium butyrate.It has previously been reported that milk formula feeding slows down intestinal maturation, including the development of intestinal motility patterns, in newborns, leading to reduced number of noradrenergic fibers in the enteric nervous system , decreasThe mechanism of butyrate’s effect on intestinal contractility at a cellular and molecular level is poorly understood. Most of the data available on butyrate are related to its effect on ileal and large bowel muscular activity, with both excitatory ,18,31,32There are indications that butyrate may act directly on smooth muscle cells ,16,33 asThus, we decided to study full–thickness intestinal segments instead of only muscle preparations as previous in vitro studies have revealed that different types of ion channels and receptors, as well as interneurons and nonmuscle cells, occur along the intestinal wall and may be crucial for the contractile response . Moreove1R, M3R, and M5R) stimulates phospholipid turnover and increases intracellular calcium levels, while activation of even-numbered MRs (M2R and M4R) inhibits adenylyl cyclase activity, thereby reducing levels of intracellular cAMP. Previous studies have shown that butyrate stimulates GH (by increasing Ca2+ and reducing GHRH-stimulated cAMP accumulation). Moreover, GH secretion is modulated by cholinergic pathways and is responsive to pharmacological suppression by muscarinic receptor blockade [Many studies have shown that butyrate enhances enteric cholinergic and noncholinergic neurons ,18,31, wblockade . We demoIt should be mentioned that, in our study, the contractility characteristics poorly correlated with changes in the intestinal muscle layer. We reported an increase in the thickness of the muscularis mucosa in the duodenum, with the lack of a similar trophic effect in the middle jejunum. Therefore, the effect of sodium butyrate on duodenal motility could be related to its direct trophic impact on the intestinal smooth muscle cells. A similar effect was observed in the muscularis layer of the duodenum in piglets receiving butyrate , but theHowever, many studies on butyrate have demonstrated an increase in the mass of the distal jejunum, ileum, and large intestine, including thickening of the mucosa and muscle hypertrophy ,20. ThisIn the middle jejunum, butyrate also increased intestinal apoptosis as well as the proliferation of crypt stem cells, which is in part consistent with the data from Guilloteau et al.  in youngThe current study provides evidence that dietary sodium butyrate has a wide spectrum of effects in the gastrointestinal tract, including increasing small intestinal contractility. Our findings indirectly provide data showing that breast milk sodium butyrate may stimulate gut motility in human infants. Supplementation of milk formula with sodium butyrate may reverse the negative effects of milk formula in the upper intestine, including the reduced motility usually associated with milk formula feeding. Immature intestinal motor patterns and enteric nervous system function have been reported in very low birth weight and preterm infants, and GI motility disorders are a common feature of infancy . Abnorma


This study aimed to summarize the computed tomography (CT) findings of PMME and differentiate it from esophageal SCC and leiomyoma using CT analysis.This was a retrospective study including 23 patients with PMME, 69 patients with SCC, and 21 patients with leiomyoma in our hospital. Qualitative CT morphological characteristics of each lesion included the location, tumor range, ulcer, enhanced pattern, and so on. For quantitative CT analysis, thickness, length and area of tumor, size of largest lymph node, number of metastatic lymph node, and CT value of tumor in plain, arterial, and delayed phases were measured. The associated factors for differentiating PMME from SCC and leiomyoma were examined with univariate and multivariate analysis. Receive operating characteristic curve (ROC) was used to determine the performance of CT models in discriminating PMME from SCC and leiomyoma.The thickness, mean CT value in arterial phase, and range of tumor were the independent factors for diagnosing PMME from SCC. These parameters were used to establish a diagnostic CT model with area under the ROC (AUC) of 0.969, and accuracy of 90.2%. In pathology, interstitial vessels in PMME were more abundant than that of SCC, and the stromal fibrosis was more obvious in SCC. PMME commonly exhibited intraluminal expansively growth pattern and SCC often showed infiltrative pattern. The postcontrast attenuation difference in maximum CT attenuation value between plain and arterial phases was the independent factor for diagnosing PMME from leiomyoma. This parameter was applied to differentiate PMME from leiomyoma with AUC of 0.929 and accuracy of 86.4%.The qualitative and quantitative CT analysis had excellent performance for differentiating PMME from SCC and esophageal leiomyoma.The online version contains supplementary material available at 10.1007/s00261-022-03556-8. Primary malignant melanoma of the esophagus (PMME) is extremely rare, accounting 0.1–0.5% of esophageal malignancies . ApproxiThe preoperative diagnostic rate of PMME was low . PatholoSince melanoma is thought to be arising from the basal layer of the epithelium and is covered by superficial epithelium, radiological appearance closely resembles non-epithelial tumors. Leiomyoma is the most common mesenchymal tumor of the esophagus . PreoperBecause of the rarity of PMME, very few studies have investigated the radiological characteristics. Previous reports found that PMME was usually polypoid, intraluminal, and nonobstructive , 17. HowThis retrospective study was approved by our institutional review board, and a waiver of informed consent was remitted.We searched for patients with surgical or biopsy-proven PMME at our hospital between January 2011 and January 2021, and 23 consecutive patients were identified. The following criteria were applied: (1) Patients with gastroscopy biopsy-proven or surgical proven PMME; (2) Patients performed chest enhanced CT examination; (3) Availability of diagnostic quality images for measuring lesions; (4) The primary site of melanoma was identified as esophagus. The exclusion criteria were as follows: (1) Patients had esophageal multiple primary tumor; (2) Patients received other treatment, such as chemotherapy or radiotherapy before CT scan; (3) Enhanced or plain chest CT data could not be obtained or the images could not be interpreted; (4) Presence of other primary sites of melanoma. In addition, 69 esophageal squamous cell carcinoma (SCC) patients who underwent surgery were included in this study. 21 patients with surgical proven leiomyoma at our hospital between January 2010 and January 2019 were identified. Finally, a total of 113 patients were obtained in this study. The complete patient enrollment process is shown in Fig. All MDCT examinations of the chest before surgery were performed using the Discovery CT750 HD scanner . Generally, the scan began at 2.0 cm above the lung apices and extended through the adrenal glands. The following imaging parameters were used: 120 kVp tube voltage; autoregulation of mA (200–400 mA) and noise index of 9; detector collimation of 64 × 1.25 mm; 0.6 s/ rotation gantry rotation speed; and helical pitch of 0.984. Axial images were reconstructed using a section width of 5.0 mm. Coronal and sagittal reformations were reconstructed using a section width of 5.0 mm. After the plain scan, the non-ionic contrast medium Iohexol  at a dose of 1.5 mL/kg body was injected at a rate of 3.0 mL/s through the median cubital vein. The enhanced scan was conducted 30 s  and 55 s (delayed phase) after the start of the contrast medium injection.The CT images were reviewed by two radiologists . The two reviewers were unaware of the final pathologic information of the patient. They assessed the qualitative characteristics of each lesion on the plain scan, arterial and delayed phases with consensus. Any discrepancy during analysis was resolved through achieving consensus by consulting a senior thoracic radiologist . Quantitative variables were recorded as the average of two separate measurements by two radiologists (Dr. Yan and Dr. Wei).The following qualitative CT findings were analyzed: (1) location ; 2) tumor range (0–1/2 and 1/2–1), for some focal lesion, double-contrast barium esophagogram was performed for assessing the tumor range; If the tumor occupied 0 to a half of the esophageal wall, the tumor range would be defined as type of 0–1/2; then, if the tumor occupied a half to whole of the esophageal wall, the tumor range would be defined as type of 1/2–1; (3) enhancement pattern (homogeneity and heterogeneity); the tumor appearing homogeneous enhancement was defined as homogeneity of enhancement pattern and the tumor appearing heterogeneous enhancement was defined as heterogeneity of enhancement pattern in arterial and delayed phases; (4) tumor air surface ; (5) necrosis in tumor ; non-enhanced area in the tumor was defined as necrosis; (6) fibrosis of peritumoral fat space ; increased focal stranding in periesophageal fat, which showed esophageal infiltration of tumor, was defined as fibrosis of peritumoral fat space; (7) ulcer of tumor  was diagnosed by the presence of air in the esophageal wall or focal necrosis from lumen to the deep level of the wall, not perforated the wall  and mean CT value of maximum axial area of tumor were obtained through ROIs of axial images. The mean CT attenuation values of tumor were recorded as the N CT value mean, AP CT value mean, and DP CT value mean, respectively. The postcontrast attenuation differences of tumor  were calculated. The ROIs were drawn to encompass the area of greatest enhancement on the maximal section in the arterial or delayed phase and copied the ROIs on the same slice in the other two phases. The type of these ROIs was circular. The maximum CT values of tumor were obtained through ROIs of axial images. The maximum CT attenuation values of tumor were recorded as the N CT value max, AP CT value max, and DP value max, respectively. The postcontrast attenuation differences of tumor  were calculated.Wall thickness of esophageal tumor was measured perpendicular to the lumen on the axial images in delayed phase. If the lumen was not visible, the maximal tumor diameter was obtained and multiplied by 0.5. Tumor length  was measured at sagittal CT imaging in delayed phase. The diameters of the short (SD-LLN) and long axis (LD-LLN) of largest lymph node were measured on the axial images in delayed phase. The regions of interest (ROIs) were manually drawn to encompass the tumor on the maximal section in axial images in delayed phase and copied the ROIs on the same slice in the other two phases. The type of ROIs was irregular. Area of tumor in largest slice . The results of the analysis of the surgical specimen served as a standard of reference.t test or Mann–Whitney test according to the condition of normal distribution. Categorical parameters were compared between PMME, SCC and leiomyoma by Chi-square test or Fisher’s exact test. Multivariate logistic regression was performed to select independent factors for discrimination between PMME, SCC and leiomyoma and to establish a predictive models. Receiver operating characteristic (ROC) curve analysis was applied to evaluate the diagnostic performance for differentiating PMME from leiomyoma and SCC, with the area under the ROC curve (AUC) calculated as well as sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy. Nomogram was constructed. Calibration curves were yielded with Hosmer–Lemeshow test. Intraclass correlation coefficients (ICCs) were determined to evaluate inter-observer agreement in terms of parameter extraction. A coefficient of 0.81–1.00 indicated an almost perfect agreement; 0.61–0.80, 0.41–0.60, 0.21–0.40, and 0–0.2 reflected substantial, moderate, fair, and poor/no agreement, respectively. Data analysis was conducted with SPSS 22.0  and R package 3.6.2 . A two-sided P < 0.05 indicated statistical significance.Differences in quantitative parameters in patients with PMME, SCC and esophgeal leiomyoma were assessed by the independent Among 23 patients with PMME, 13 patients underwent thoracotomy with tumor enucleation. Other patients with biopsy-proven PMME were not operative candidates because of the health issues or unresectable tumors. The clinical characteristics of the patients are summarized in Table P < 0.001) (Table Tables 1) Table .Table 2UTable The multivariable analysis showed that ΔAP-N CT value max was an independent factor for differentiating PMME from esophageal leiomyoma. The performance of this parameter larger than cutoff value of 16HU for diagnosing PMME from leiomyoma was well with AUC of 0.929. Detailed information on the performance of the combined CT models for distinguishing PMME from SCC and leiomyoma is shown in Table To provide clinicians with an easy tool, a nomogram based on quantitative and qualitative CT parameters, including thickness of tumor, range of tumor, and CT mean value of AP, was developed Fig. . The calThe PMME arose from the melanocytes in the basal layer of squamous epithelium. Tumor cell nests could be seen in the basal layer of squamous epithelium at the junction of tumor and normal esophagus, suggesting that the tumor originated from esophagus. Regarding its gross appearance, PMME often presented a broad base polypoid appearance which often grew expansively in submucosa layer and rarely eroded the entire mucosal epithelium Fig. . The surMicroscopically, PMME was mainly composed of sheet of melanoma cells with rare stroma reaction. The boundary of PMME between tumor area and peripheral normal tissue was usually smooth and clear with involving a small range of esophageal wall Fig. . The infFocal necrosis was a common morphological feature in both PMME and SCC. For blood supply, small vessels were easy to be observed in tumor sheet of PMME, but only presented in the fibrous stroma around the tumor nests for SCC. Interstitial vessels in PMME were more abundant than that of SCC in CD34 immunohistochemical staining Figs. . RegardiGrossly, leiomyoma appeared as a well-defined mass in the esophageal wall and had a solid, grayish white appearance on cross section. Ulceration of the overlying mucosa was uncommon. Microscopically, leiomyoma had the usual characteristics of a benign smooth muscle tumor with a clear boundary and stain for muscle markers such as desmin and smooth muscle actin. Local resection or enucleation was usually successful. Blood vessels of the leiomyoma were fewer than that of squamous cell carcinoma and melanoma Fig. .Fig. 643The treatment of choice for PMME was surgical resection with dissection of the lymph nodes . BecauseIn this study, we found that PMME tended to appear intraluminal expansively growth pattern, which meant a broad base polypoid appearance mass protruding to the lumen with well-circumscribed boundary and smooth surface. In contrast, diffuse thickening and infiltrative growth pattern were more likely to occur in SCC lesions. It was consistent with other reports , 17. PreOur study showed that the thickness of PMME was higher than that of SCC and was one of the independent parameters for diagnosing PMME. The expansive growth type may result in the high size in PMME. The origination from basal layer, and expansive growth type in PMME may contribute to less esophageal symptoms and result in the higher size at initial diagnosis. Significant differences in CT value-related parameters were AP CT value mean and max between PMME and SCC in univariable analysis. We also found that PMME more often presented obvious enhancement in arterial phase, and SCC was prone to show progressive enhancement and enhanced peak in delayed phase. Tang et al. also found that enhanced CT scanning revealed obvious lesion enhancement, suggesting a rich tumor blood supply . The othLeiomyoma was the most common benign tumors of the esophagus . The majEven if the endoscopic biopsy was used, the preoperative diagnostic accuracy of PMME was only about 80% . SometimIn terms of clinical use, the radiologists could consider the result of diagnostic models in diagnosing PMME according to the probability of PMME from 0 to 1. The parameters of an esophageal tumor were applied to the two models, respectively. If there was a difference in diagnosis of PMME between the two models, the subjective diagnosis was needed to correct the diagnosis by combining radiological findings and clinical features. The differentiation between SCC and esophageal leiomyoma was evaluated by radiologists, considering the clinical and radiological features. When esophageal tumors were difficult to diagnose by radiologists assisted by our models, we recommend further evaluation with endoscopic ultrasound (EUS) and fine-needle aspiration (FNA) to look for the presence of PMME. When our CT analysis suggested a diagnosis of PMME for the esophageal lesion, but the histological diagnosis cannot identified in the initial biopsy, the immunohistochemical analysis of biopsy specimen or a deeper esophageal biopsy should be performed to obtain an accurate diagnosis.There were several limitations in this study. First, it was a single-center retrospective study, and sample ratio might be biased. Second, because of the rarity, our study only included a small number of patients of PMME; a much larger database from multicenter with considerably larger sample was needed to validate the robustness and reproducibility of our result. Third, not all patients received surgery and the resected specimens were not allowed for pathological analysis because the PMME had aggressive biological behavior and these patients without receiving surgery were diagnosed late. Fourth, among these non-epithelial neoplasms, esophageal leiomyoma was the most common mesenchymal tumor of esophagus, unlike in the gastrointestinal tract, where GISTs predominated; the other non-epithelial neoplasms were extremely rare . So, we Comparing with SCC, PMME was prone to manifest expansive growth pattern, more arterial enhancement, larger tumor thickness, and less infiltrative range. This CT model might hold promise in discriminating PMME from SCC. Comparing with leiomyoma, PMME showed more obvious heterogeneous enhancement pattern, the presence of malignant lymph nodes, necrosis in the tumor, and non-smooth surface of tumor. These CT models might hold promise in discriminating PMME from SCC and esophageal leiomyoma. Although PMME was less common than SCC, radiologists should be familiar with the imaging and pathologic features of PMME as well as their malignant behavior and appropriate patient management.Supplementary file1 (TIF 1710 KB) Fig. S1 Nomogram based on ΔAP-N CT value max for differentiating PMME from esophageal leiomyoma and the corresponding calibration curves. (a) The developed nomogram. (b) Calibration curves depicted the calibration of nomogram in terms of the agreement between the predicted probability of PMME and actual outcomes of the PMME. The y axis represented the actual probability of PMME. The x axis represented the predicted probability of PMME. The red line represented a perfect prediction by an ideal model. The blue line showed the performance of the CT model. The blue line was closer to the red line, which suggested a better prediction.Below is the link to the electronic supplementary material.


Bread wheat, one of the largest broadacre crops, often experiences various environmental stresses during critical growth stages. Terminal drought and heat stress are the primary causes of wheat yield reduction worldwide. This study aimed to determine the drought and heat stress tolerance level of a group of 46 diverse wheat genotypes procured from the Australian Grains Gene Bank, Horsham, VIC Australia. Two separate drought stress (DS) and heat stress (HS) pot experiments were conducted in separate growth chambers. Ten days after complete anthesis, drought (40 ± 3% field capacity for 14 days) and heat stress (36/22 °C for three consecutive days) were induced. A significant genotype × environment interaction was observed and explained by various morpho-physiological traits, including rapid, non-destructive infrared thermal imaging for computational water stress indices. Except for a spike length in DS and harvest index in HS, the analysis of variance showed significant differences for all the recorded traits. Results showed grains per spike, grains weight per spike, spike fertility, delayed flag leaf senescence, and cooler canopy temperature were positively associated with grain yield under DS and HS. The flag leaf senescence and chlorophyll fluorescence were used to measure each genotype’s stay-green phenotype and photosystem II activity after DS and HS. This study identified the top ten best and five lowest-performing genotypes from drought and heat stress experiments based on their overall performance. Results suggest that if heat or drought adaptive traits are brought together in a single genotype, grain yield can be improved further, particularly in a rainfed cropping environment. Being the staple food of over 35% of the world’s population , bread wAmong various environmental constraints, drought and heat stresses are two main factors that severely restrain crop growth and productivity worldwide . EstimatWheat is highly sensitive to heat stress during critical phenophases . The croc), crop water stress index (CWSI), and canopy temperature depression (CTD)) for drought and heat stress study in wheat. The Tc and CWSI fluctuate with the surrounding environment and negatively correlate with the final grain yield [Drought and heat stress accelerates maturity and reduces leaf colour and chlorophyll contents, leading to premature plant senescence . Photosyin yield .The screening of diverse germplasm is critical in identifying drought or heat-stress-tolerant and susceptible genotypes. This approach requires the utilization of appropriate phenotyping methods, identification of the accurate developmental stage most prone to stress events, and relevant traits of interest for a stress tolerance study . Based ob1 = G6–G15, b2 = G16–G25, b3 = G26–G35, b4 = G36–G46). The checks were replicated in each of the four blocks, while test genotypes were grown only once in the experiment. The repetition of checks enables the estimation of the error variance and blocking effects for statistical inference, resulting in more precise estimates of the treatment comparison of interest. The position of the test genotypes and the checks were fully randomized in each block.Forty-six wheat genotypes (G1–G46), including five checks (G1–G5), were procured from the Australian Grains Genebank, VIC Australia . A non-rThe genotypes were sown in polypropylene square-shaped nursery pots  with five biological replicates in plant growth chambers (PGR15) at the Dookie Campus, The University of Melbourne, Australia  from October 2018 to February 2019. Initially, eight seeds of each genotype were sown per pot and later thinned to five after ten days of sowing for optimum plant growth. The three harvested seedlings were used to destructively measure the early vigour of wheat genotypes.−1 available N, 210 mg kg−1 Colwell-P, and 660 mg kg−1 available K. Standard agronomic practices and appropriate fertilization were implemented to minimize yield limitations.For the experiment, the soil was collected from the upper 15–20 cm layer of a cultivated field from the mount major lane wheat paddock of Dookie Campus and sieved well with 4 mm mesh to remove plant debris and stones for homogenization. Each pot was filled with 6 kg of sieved soil. The soil was red-brown Dookie clay loam, classified as Gowangardie loam . The soi−2 s−1 above the plant canopy. The relative humidity (RH) of the PGR15 was set to 65–70% [For ideal growing conditions, the PGR15 were programmed at 24 ± 1 °C and 18 ± 1 °C 13/11 h) for the day and night period [ h for tho 65–70% .The genotypes were individually monitored for their anthesis time and split into three groups according to the early, mid, and late maturity life cycle. Before drought or heat stress treatment, each group was monitored to determine when their primary tiller reached the first phase of grain filling, ten days after complete anthesis (Zadoks growth stage 75) ,29. At tFor DS treatment, water stress with 40 ± 3% pot field capacity (FC) was imposed on E1 pots for an entire 14-days period. Before the start of the DS, irrigation was slowly reduced for five days to attain 40 ± 3% FC . The RH Following Rebetzke and Richards  method, 0). After the initial weight, the leaves were immediately shifted to a controlled growth room maintained at 20 °C with 50–60% of RH and 250–300 µmol m−2 s−1 of continuous light. The leaf weights were recorded after four hours (FW4), eight hours (FW8), and finally after drying (DW) at 65 °C for 48 h. The excised leaf water loss after four hours, eight hours, and from four to eight hours was estimated as a percentage of water loss per unit of initial water contents as follows.The excised leaf water loss (ELWL) of each genotype was estimated using the CLARKE McCAIG  method. The sections of leaves (8–10 cm long) were taken on the last day of the DS treatment. The fresh weight of leaf samples (FW) was determined instantly after excision, followed by soaking in distilled water in plastic tubes for 12 h at standard room conditions. The hydrated samples were then taken out of the distilled water, gently dried with tissue paper, and reweighed for the turgid weight (TW). The leaf samples were then oven-dried at 70 °C for 48 h for their dry weight (DW). Following Barrs and Weatherley , the RWCv/Fm) of each genotype was measured with a portable LED modulation fluorescence system . Minimal chlorophyll fluorescence (Fo) was determined in dark-adapted leave discs (30 min) under low modulated actinic light (>0.04 μmolm−2 s−1) followed by a 1-s saturated white light pulse (>3000 μmolm−2 s−1) in the discs of same leave to estimate maximal chlorophyll fluorescence (Fm). The quantum yield of photosystem II for dark-adapted leaves was calculated as reported by Carvalho and Amâncio [Before the end of the DS treatment, the chlorophyll fluorescence (F Amâncio .(5)FV/F1 and T1, respectively) were recorded with the TPS AQUA-CPA Cond/Salinity meter. After the initial readings, test tubes were put together for a 20 min autoclave at 120 °C and 100 kPa for a second conductance reading . The thermostability of the cell membrane was calculated as a relative cell membrane injury percentage  using the following formula.The thermostability of the cell membrane was evaluated by following the Martineau et al.  method. The flag leaf senescence (FLS) of all studied genotypes was estimated, starting from one week after heat stress to physiological maturity. The loss of green leaf area (SPAD readings) after HS and during the recovering period in an index of the FLS. A Minolta SPAD-502 plus chlorophyll meter was used to measure the FLS of all five plants of each genotype. The measurements were carried out at weekly intervals for six weeks. Around 1 cm from the edges, three readings were taken downside of the leaf from the base, centre, and tip.m) was calculated by adapting ‘Equation (5)′ from the Borrell et al. [a and SPADm are SPAD readings taken after weekly intervals of heat stress and physiological maturity, respectively.The relative rate of flag leaf senescence at physiological maturity  and crop water stress index (CWSI) [c is the plant canopy temperature (°C), Twet is the reference temperature (°C) of a non-stressed plant, and Tdry is the reference temperature (°C) of the stressed plant. Twet and Tdry are empirical reference temperatures estimated through the statistical frequency analysis of IRTI.Before the end of the drought and heat stress, infrared thermal images (IRTI) of each genotype were taken using a thermal camera . At 30 °C, the spatial resolution and thermal sensitivity of the camera was 0.47 milliradians and <20 mK, respectively. The object temperature measurement range of the camera is −40 °C to 150 °C. Each Pixel of IRTI holds a 16-bit thermal value in a °C unit. The thermal images were taken in standard room conditions to avoid plant canopy temperature acclimatization with the surrounding environment. To calculate the canopy temperature (Tx (CWSI) ,38, the x (CWSI) ,37,39 thc and empirical reference temperature data, an Infrared index (Ig) of crop stress indices, which is proportional to the stomatal conductance (gs) and water vapour transport (gw), was estimated using the relationship proposed by Jones et al. [aw is the resistance to gw in the boundary layer/air, “s” is the slop of the curve relating temperature with saturating vapour pressure, γ is the psychrometric constant, and rHR is the parallel leaf resistance to the heat transport and radiative heat loss.Using Ts et al.  and Fuens et al. .(9)Ig=Tc from mean environmental temperature (Ta) and it is positive when the plant canopy is cooler than the surrounding environment.Canopy temperature depression (CTD) was also determined from IRTI analysis and calculated by subtracting the TThe agronomic traits, grain yield, and yield components were estimated for both experiments. The days to 50% anthesis (D50A) was recorded as the total day count from sowing to extrusion of anthers in more than 50% spikes (Zadok stage 69) . Plant h2p), genotypic variance (δ2g) and environmental variance (δ2e), phenotypic, genotypic, and environmental coefficient of variance , category of genotypic and phenotypic coefficient of variance, genetic advance (GA), and genetic advance as percent over mean (GAM) were computed from an object of class of “augmentedRCBD” package.An analysis of variance (ANOVA) was performed separately for both experiments using the “augmentedRCBD” package of R statistical software ,44. For c, CTD, Ig, Tdry, Twet, and CWSI [The thermal images data was extracted using MATLAB code scripted in MATLAB R2017b  and Imagand CWSI  and analand CWSI ,46. The and CWSI .p < 0.05) for all the wheat genotypes and revealed associations among EVG components (The genotypic effects for EVG were significant (mponents . Genotypp < 0.05). Except for spike length in DS and harvest index in HS, highly significant differences were observed for all the measured traits within the main effects of blocking on treatment and their interactions  were observed across all the wheat genotypes for the mean values of recorded traits under DS and HS. g, which indicates good stomatal conductance activity of these genotypes for keeping lower Tc  under DS. Genotypes like WH147, Excalibur, RAC875, and ECH957 (top-performing genotypes) had the maximum spike fertility compared to CM59443 and TR274 (low-performing genotypes). Based on ranking criteria, the genotypes ECH957, RAC875, WH147, Drysdale, Excalibur, M723, RAC622, RAC704, AL24, and BR670 were considered drought-tolerant genotypes, while the genotypes Kukri, ECH952, TR165, TR274, and CM59443 were considered drought susceptible genotypes , ECH957, RAC875, RAC622, CM61981, WAGGA51, and Excalibur had minimum values under HS. Based on ranking criteria, genotypes ECH957, Axe, TR188, RAC875, RAC622, CM61981, WAGGA51, and Excalibur were considered heat-tolerant genotypes, while Kukri, CM59443, TR274, CM78566, and WH147 were considered heat susceptible wheat genotypes  was observed on the flag leaf chlorophyll content in all the genotypes  from the standardized genotypes-by-traits data matrix explained a total cumulative variance of 61.8% for the first two principal components (PC1 = 34.9% and PC2 = 26.9%) for the E1 and 70.1% (PC1 = 39.1% and PC2 = 31%) for the E2 A,B. Tablc due to their obtuse angles with grain yield under DS  due to their obtuse angle with Tc  are shown in dry, Twet, and Tc showed a strong positive correlation among themselves. In contrast, CTD showed a strong negative correlation with Tdry, Twet, and Tc. Similarly, ELWL0–4h, ELWL4–8h, and ELWL0–8h showed weak but significant correlation with spike fertility and moderate but significant correlation with days to leaf rolling. For HS, grain yield had a significant positive correlation with spikelets per spike, harvest index, grains per spike, spike length, spike fertility, and grains weight per spike. Results showed that CTD showed a strong negative correlation with Tdry and Twet compared than CWSI and Tc.Correlation matrix plots describing the degree of correlations between recorded traits  compared with ECH961 and K14077, which were grouped at different leaf heights and clade within the same cluster .Germplasm screening for drought and heat stress in a controlled environment is an effective strategy for selecting plants with desirable traits for future research . Early v2p classification into δ2g and δ2e is not enough for complete information on any source material. Like the coefficient of variation, genetic advance over mean can also be classified into low (<10%), medium (10–20%), and high (>20%) categories [The ANOVA  and genetegories . Higher tegories . It alsotegories . This meDifferent yield components have also been used to screen genotypes for drought and heat stress. Severe DS and HS tended to accelerate plant growth and senescence earlier, resulting in lower grains weight per spike due to the insufficient mobilization of metabolites and shorter grain filling duration ,58. The The evaluation of ELWL and RWC as screening techniques in drought stress has been associated with improved morpho-physiological and anatomical traits in wheat . The usec under DS or HS [c were observed across the genotypes. Results showed that the use of Tc to assess the level of water stress on the genotypes depends upon stomatal conductance  with higher positive CTD values imply that they had managed Tc to a metabolically suitable level even under soil moisture deficit conditions. A cooled canopy ultimately leads to higher photosynthesis and more above-ground dry matter accumulation. This might be due to the effectiveness of the rooting system of responding genotypes in extracting soil moisture to maintain Tc [A canopy temperature depression measures the effectiveness of the canopy cooling capacity in maintaining the Tstresses ,68. In tg and gs while studying the water status of different horticultural crops such as grapevines [g is linearly related to gs, Ig is primarily attributed as a water stress indicator [g values across the genotypes (s under DS or HS and (ii) varying water requirements of the genotypes due to differences in the developmental stage. Plants respond rapidly to regulate Tc in a changing plant water status by opening and closing stomata through leaf gas exchange fluxes. The modelling of Ig and CWSI shows that both indices have similar discriminative power. Due to a linear relationship with gs, Ig data recording is recommended, at least at the leaf level [Previously, a good association was found between Iapevines , olive oapevines , and fieapevines . Althougndicator . In the enotypes , and thiaf level .2), and it shows the importance of PCs for a given variable. The cos2 represents the contribution of PCs to the squared distance of the variable on the factor map. Therefore, the value of cos2 from the origin can help find PCs that are important to interpret both supplementary and active observations [2 points approximate the row information (genotypes). Genotypes with a higher cos2 value on either side of the PCs indicate the quality representation of phenotypic components on the factor map [2 points shows the magnitude of similarity and dissimilarity between the corresponding genotypes. When two vectors have higher sharp angles with each other, the correlation between them is weak, and they are independent of each other. The same direction and smaller acute angles between dimension vectors indicate an inter-relationship among the trait variable concerning discriminating wheat genotypes.The principal component analysis is a dimensionality reduction multivariate technique that extracts the relevant information from the original data and allows the visualization of variables (loadings) and objects (scores) on the factor map . In the rvations . The vecctor map . The disc, CTD, Ig, Tdry, Twet, and CWSI) and other physiological measures indicated that most of the traits expressed differentially among tolerant and susceptible genotypes under DS or HS conditions. This study reveals that with other morpho-physiological attributes, IRTI proved to be an important constructive technique for a spatially explicit assessment of plant water status under DS and HS. As a high-throughput phenotyping approach based on plant–water status, IRTI also provides a powerful insight to crop breeding programs for evaluating a large group of genotypes for their adaptability to drought or heat stress in a typical field condition. The stay-green trait provides valuable information for the diagnosis of plant photosynthesis status and to assess the health of the other plant physiological systems. Results from this study indicated that genotypes identified as drought and heat-tolerant  or susceptible  based on a set morpho-physiological measures including IRTI could potentially be used as a useful genetic resource in future drought and/or heat stress related pre-breeding and breeding programs. However, the performance of these selected genotypes requires further evaluation to assess their growth and yield performances in a natural field environment under drought or heat stress conditions.The screening of wheat genotypes for terminal drought and heat stress is valuable for future research experiments that integrate drought or heat stress tolerance studies. Significant genotypic variation among IRTI water stress indices (T


In this study, native lotus seed flour (N-LSF) was modified by different methods, namely, partial gelatinization (PG), heat–moisture treatment (HMT), or pullulanase treatment (EP). Their composition, functional properties, starch composition, and estimated glycemic index (eGI) were compared. PG contained similar protein, soluble dietary fiber, and insoluble dietary fiber contents to N-LSF, while those of HMT and EP differed from their native form. PG increased rapid digestible starch (RDS) but decreased resistant starch (RS); while HMT and EP increased amylose and RS contents to 34.57–39.23% and 86.99–92.52% total starch, respectively. Such differences led to the different pasting properties of the modified flours rather than PG, which was comparable to the native flour. HMT had limited pasting properties, while EP gave the highest viscosities upon pasting. The eGI of all samples could be classified as low (<50), except that of PG, which was in the medium range (60). It was plausible that lotus seed flour modified either with HMT or EP could be used as carbohydrate source for diabetes patients or health-conscious people. Diabetes mellitus (DM) is one of the metabolic diseases characterized by high fasting blood glucose levels, of which the prevalence and incidence are currently increasing glob-ally. It has been reported in 2021 that 537 million adults around the world are living with DM, and the number estimated in 2030 will rise to more than 643 million [Nelumbo nucifera), is a rich source of starch, which provides carbohydrates as a nutrient and energy source for the human body. Many studies have reported that total starch content in lotus seed can reach over 60% on a dry basis [Bifidobacterium adolescentis growth and produce short chain fatty acids when tested in the simulated conditions of the human digestive system. Thus, lotus seed can be used as a prebiotic. In addition, lotus seed starch possessed low swelling power, and consequently high pasting and gelatinization temperatures, though the gelatinized granules are highly susceptible to enzymatic digestion. It also showed higher gel syneresis and higher ratio setback to peak viscosity during pasting, suggesting a stronger tendency for short-term retrogradation [Lotus seed, particularly of sacred lotus  were purchased in four batches from a local farm located in Nakhon Sawan province, Thailand during July 2020 to September 2020. Once received, the seeds were sampled for moisture analysis, vacuum-packed in polyethylene plastic bags and stored at room temperature until being used for flour preparation. Moisture content of the lotus seeds ranged from 6.5–7% w/w. Prior to flour preparation, lotus seeds were soaked in deionized (DI) water  overnight, peeled, removed the embryos by hands, and dried in a hot air oven at 40 °C for 16–18 h to obtain dried lotus seeds with the final moisture content of 7–8% w/w. Native lotus seed flour (N-LSF) was prepared by grinding the dried seeds using a grinder mill machine, followed by sieving through a 100-mesh sieve. The flour was packed in polyethylene plastic bags and kept at room temperature until being used. The production yield of N-LSF from dried lotus seeds was 80–82%.Sun-dried seeds of sacred lotus  and kept under refrigeration at 4 °C for 12 h. They were parboiled at 75 °C for 10 min to achieve partial gelatinization of starch. Then, the parboiled seeds were dried at 60 °C in a hot air oven until the moisture content was 10–12% w/w. After drying, the seeds were ground and sieved through 100-mesh sieve to obtain partially gelatinized lotus seed flour (PG). The samples were packed in polyethylene plastic bags and kept in a desiccator. The yield of production of PG from dried lotus seeds was up to 80%.Dried lotus seeds were soaked in DI water  was prepared by slightly modifying the method of Whantongkhum and Suwannaporn . DI watew/v) of N-LSF in DI water was prepared. The pH of the slurry was adjusted to 4.5 with 0.1 M sodium acetate buffer solution pH 4.5. Pullulanase  was added to the slurry at an enzyme-to-substrate ratio of 20 NPUN/g dry flour. Enzymatic treatment was performed at 55 °C for 6 h in a shaking water bath. The treated slurry was then heated at 70 °C for 10 min to stop the enzyme reaction. Sample was centrifuged at 4600× g for 10 min, and the precipitate was washed twice with DI water. The precipitate was collected and dried at 45 °C in a hot air oven until the final moisture content reached 10–12% w/w to obtain the enzymatically modified flour (EP). The recovery of EP from N-LSF was about 70%.Enzymatic modification of lotus seed flour was performed using pullulanase according to the method of Wattananapakasem et al.  with sliChemical composition of native and modified lotus seed flours was determined according to the AOAC Official Methods . Moistur2 + 2 g KI in DI water, 800 μL). A final volume was then made up to 12 mL with DI water. The sample was immediately mixed and chilled in the refrigerator for 20 min before test. Amylose content was determined using 96-well UV visible microplate reader spectrophotometer  at the wavelength of 620 nm. The absorbance values were converted to percent of amylose by standard curve of amylose from potato .Amylose content was determined using the iodine absorption method  with sliRapid digestible starch (RDS) and slow digestible starch (SDS) contents were analyzed based on in vitro starch digestibility procedure . Digestag for 10 min. The residue as washed with 10 mL of DI water and recentrifuged at the same condition. Then, 3 mL of DI water and 3 mL of 4 M KOH was added and the mixture was incubated at room temperature for 30 min. After that, 5.5 mL of 2 M HCl, 3 mL of 0.4 M sodium acetate buffer pH 4.75 and 80 μL (3.26 U) of amyloglucosidase in 0.4 M sodium acetate buffer pH 4.75 were added, followed by vortexing. The mixture was incubated at 60 °C for 45 min in a shaking water bath. Next, it was centrifuged at 4600× g for 10 min to collect the supernatant. The residue was washed with 10 mL of DI water and centrifuged again. The supernatant from the first and second extractions were combined for the measurement of glucose content using glucose oxidase–peroxidase kit GOPOD reagent enzymes .Resistant starch (RS) content was analyzed based on an in vitro starch digestibility procedure  with slig for 10 min. The supernatant was carefully pulled out using a syringe, dried for 24 h at 105 °C and weighed. The precipitates were immediately weighed. Solubility and swelling power were calculated according to the following equations:Solubility and swelling power and of samples were determined according to method of . Flour s® v.2.2, Newport Scientific).Pasting properties of samples were investigated according to the AACC method 76–21.01  using a g for 10 min to separate precipitate for RS analysis. The supernatant was further added with 0.4 M sodium–acetate buffer  and amyloglucosidase from Aspergillus niger . Samples were incubated for 45 min at 60 °C. Glucose concentration in sample was then measured using glucose oxidase–peroxidase kit GOPOD reagent enzymes (Megazyme). Rate of starch digestion was expressed as a percentage of glucose at different times . The hydrolysis index (HI) was initially determined using area under the curve from GraphPad Prism 7 software  and using white bread as a reference food. From the linear relationship between hydrolysis index and glycemic index, estimated glycemic index (eGI) was calculated according to the equation:The rate of in vitro starch hydrolysis was analyzed according to the method recommended by . BrieflyIn addition, glycemic load (GL) of the flour samples was determined from eGI and the amount of available carbohydrate in a common serving, i.e., 30 g (1/4 cup). Available carbohydrate was calculated by subtracting total dietary fibers from total carbohydrates. GL was calculated using the following equation:p < 0.05).Statistical analyses were performed using the IBM SPSS Statistic 19 . All results were reported as mean and standard deviation of three replicates, except that chemical composition was reported as mean of duplicate analysis. Differences between lotus seed flour and its modified samples were determined using Analysis of Variance with Tukey’s post hoc test . Protein content of N-LSF was 26.79% on a dry basis, which was the highest among other samples. Carbohydrate was the major composition of N-LSF, accounting for 67%, while fat and ash contents were only about 2 and 4%, respectively, on a dry basis. Partial gelatinization and pullulanase treatment caused the lotus seed flour to have slightly less protein than the native flour, while heat–moisture treatment decreased the protein content in lotus seed flour to about 12% dry basis. The preparation of HMT involved soaking of the flour, which probably led to the loss in soluble protein by leaching. Denaturation of protein during the heat moisture treatment may also have contributed to the decrease in protein content of HMT. This result is in agreement with work of Jia et al. [Moisture content of native lotus seed flour was 7.92% .48% w/w . All lota et al.  that theIt is noticeable that protein content of N-LSF was significantly higher than other conventional flours, such as rice (9%), corn (8%), wheat (15%), and potato (9%) ,38. MoreFat content of PG and HMT was similar to that of native flour (about 2% dry wt) but EP contained slightly lower fat than the others. This was because all modification methods did not involve any hydrophobic solvent that could lead to the change in fat content. Similar ash content of 4.3% was also observed for N-LSF and PG, while HMT and EP contained less ash at 2.33 and 1.06%, respectively. It was plausible that the decrease in ash content of HMT and EP was caused by leaching of minerals into water during those modification processes. The total carbohydrates content of HMT and EP was higher than N-LSF and PG. This finding could be explained by the lower protein and ash contents of HMT, and the lower fat and ash content of EP, when compared with N-LSF and PG.N-LSF contained 8.36 and 3.59% of soluble and insoluble dietary fiber, respectively, on a dry basis. Slightly lower amounts of both types of dietary fibers were found in PG. Significant alteration of dietary fiber content was observed in HMT and EP. Heat–moisture treatment gave lotus seed flour with lower soluble fiber but higher insoluble fiber content. An opposite trend was observed for EP, of which most of the dietary fiber was soluble fiber and there was only <0.10 g of insoluble fiber in 100 g dry wt. Soluble components including soluble dietary fiber might have lost upon the preparation steps of HMT. In addition, it was possible that heat–moisture treatment led to the denaturation of protein in the lotus seed flour; thus, the denatured protein could form a layer on the surface of starch granules. Consequently, the protein-coated starch granules were inaccessible to the enzymes used for dietary fiber analysis and became less soluble in water. Dupuis et al.  demonstrBased on the colorimetric iodine method, the percentage of amylose in N-LSF was 24.30% on a dry basis . It has The enzymatic digestibility of native and modified lotus seed flours was investigated. RDS, SDS, and RS, which were classified consecutively by reaction times, represented the three different starch materials found in lotus seed flour samples . N-LSF cSolubility, also known as water-soluble index, of native and modified lotus seed flours were studied at 55, 65, 75, 85, and 95 °C, and the results are shown in The swelling power, which indicates the amount of water that can penetrate into and be adsorbed by the flour, of different lotus seed flour samples is presented in Although swelling power is known to be harmonious with solubility ,18, therPasting properties are associated with the rigidity or swelling potential of starch granules and the leaching of amylose molecules. The results showed that all pasting parameters of N-LSF were relatively closed to those of native lotus seed starch in the literature . DiffereThe pasting properties, particularly peak viscosity, peak time and pasting temperature, of native and modified lotus seed flours correlated with their swelling power . Flours For EP, the short linear amylose chains, which were abundant in EP , might lIn this study, starch hydrolysis was carried out in vitro to simulate the in vivo digestive characteristics of lotus seed flours and to estimate their glycemic indices. As shown in Starch modification based on partial gelatinization, heat–moisture treatment, and pullulanase enzyme treatment significantly altered the composition and properties of lotus seed flour to different extents. Partial gelatinization did not affect the chemical composition and pasting properties of the flour but largely increased the digestibility of starch. Heat–moisture treatment resulted in the modified lotus seed flour with less protein but more amylose, insoluble fiber, and RS, which restricted its swelling and pasting behavior while maintaining the low GI value of the native flour. Treatment with pullulanase altered the properties of lotus seed flour in a similar manner to heat–moisture treatment, but the enzyme-modified flour exhibited more rapidly pasting and much larger changes in viscosity during pasting cycle than both the native and other modified flours. Based on the obtained results, heat–moisture treatment could be a potential modification method for producing low glycemic lotus seed flour that could be applied in food product without affecting the consistency of food. On the other hand, pullulanase treatment could improve the thickening effect of lotus seed flour while maintaining the low glycemic value.


Solanum lycopersicum) cv. Momotaro seedlings were transplanted in a randomized complete block (RCB) manner with six plants/treatment, and an overall 36 plants in 18 pots (2 plants/pot). The experiment involved varying SAL treatment  and FT treatment (NT: no thinning and 3FT: three-fruit treatment). The tomato growth, yield, and WUE were significantly suppressed with increasing SAL. In comparison, FT treatment had less effect on tomato growth and water consumption. Either SAL stress or FT treatment significantly improved fruit quality. The combined treatment proved better than single treatment of either SAL stress or FT, avoided the subsize fruit following SAL stress treatment, reduced fruit cracking found with FT treatment, and greatly improved fruit quality. The SAL thresholds of WUEs in relation to biomass, yield, and marketable yield were approximately 3.0 dS m−1 under these soilless conditions. Path analysis showed that biomass and water consumption were important indexes affecting yield. Logistic equation fitting showed that SAL stress tended to inhibit and delay plant growth; however, FT tended to advance and shorten the period of plant growth.This was an experimental investigation of the combined treatments of salinity (SAL) stress and fruit thinning (FT) on the growth, yield, fruit quality, and water use efficiency (WUE) of tomatoes with non-soil cultivation. The experiment was carried out in a plastic tunnel, Japan. Tomato ( Global urbanization was accelerating, and it was reported that 60% of the global population will live in urban areas by 2030 . People Solanum lycopersicum) is one of the most important vegetable plants in the world. Global production exceeds 180 million metric tons, with China and the USA as the leading producers in 2019, and production in China accounts for 20% of the world (−1)  , and grerld (−1) , 17 and rld (−1) , and plarld (−1) . The decrld (−1)  and in frld (−1) . Howeverrld (−1) , 26.The studies also showed that yield, fruit quality, and fruit size were affected by numerous reasons, such as plant community , 28, fruThe environmental thermal status remarkably affects the tomato physiological process , 37. PlaAccording to the above explanation, soilless cultivation is widely used in the home garden because of its convenience and efficiency. SAL stress reduces tomato fruit size and marketable yield, but increases fruit quality. FT increases fruit size and quality, but increases the risk of fruit cracking and so reduces marketable yield. The above two cultivation methods show contradictory advantages and disadvantages for tomato growth. Therefore, it is necessary to determine an appropriate combined treatment of SAL stress and FT for tomatoes. However, the previous research primarily highlighted SAL stress or FT. There has been few considerations of combined treatment, and few relevant studies have used soilless cultivation. Consequently, our research aimed to explore  the combThe assay was carried out inside a plastic tunnel  at the Experiment Farm of Gifu University, Japan . The experiment duration was for 12 weeks from April to July 2018, with average daytime and nighttime temperatures of 28.1 and 20.4°C, respectively, and corresponding average humidity of 51.5 and 79.7%.Solanum lycopersicum) cv. Momotaro plants were grown via a purchasable hydroponic kit , which includes a nutrient solution bucket, a plant table, and a plant bowl  and 4.5 dS m−1 (serious stress) were employed herein. A total of six treatments were utilized, with three electroconductivity results  of SAL treatments and two levels (NT: no thinning and 3FT: three fruits) of FT treatments  when the plant was in the 3rd flower cluster stage. Considering the variation in the climate environment in the house, the location of the pots was changed after irrigation. The cultivation liquid was renewed once every 2 weeks. SAL stress began on May 22 and the first fruits were pruned on May 24.−1) of each pot was determined via gravimetry, with the following calculation:The transpiration , W′d denotes the weight of the replaced liquor (g), and W″i denotes the weight of watering (g). Watering was carried out supplementing cultivation liquid once every 1 or 2 days. The overall TP of each pot was divided between the two plants in every pot as per their eventual phytomass, and the overall TP of each plant is deemed as the plant's actual TP. The electroconductivity of every pot was surveyed posterior to watering. The solution or NaCl was supplemented when the liquor electroconductivity diverges from the normal values of 3.0 ± 0.2 and 4.5 ± 0.2 dS m−1.in which TP denotes the transpiration when the liquor is renewed (g), Wvia a SPAD chlorophyll meter (Minolta). A linear association existed between SPAD results and extracted leaf chlorophyll  of every plant was identified posterior to the assay. Leaf chlorophyll was surveyed once a week orophyll , 40; heny weight .−1), the number of flowers, and the number of fruits on every plant were surveyed in harvesting periods. The harvested fruits were divided into marketable or unmarketable. Marketable ones were divided as per size (≥30 mm) and appearance (no cracking). The sizes were measured by vernier caliper. The marketable fruit number and the marketable yield on every plant were also surveyed. The fruit set  and marketable fruit set  on every plant were calculated.Red fruits were harvested two times a week in harvesting periods. The yield  denotes grams of biomass generated per kg of water; WUEy (g kg−1) denotes grams of yield generated per kg of water; and WUEmy (g kg−1) denotes grams of marketable yield generated per kg of water.in which WUEvia a squeezer, and the sugar content (%) and acid content (%) were determined with a Pocket Brix-acid meter . Moreover, taste index was computed via the formula of Hernández-Suárez et al.  and the yield not only displays the correlation between these factors, but also presents the direct and indirect effects of growth factors on yield , 44. In A logistic equation was used to quantify the effects of SAL and FT on the growth of tomatoes, which is expressed below , 45:y denotes the dependent growing parameter or plant biomass cumulation (g plant−1); k denotes the top asymptotic line reflecting the superior limit of plant growth; a and b denote quotient of the preliminary stage and accretive rate, separately; and t denotes the independent runtime (°C d).in which Feature parameters of the logistic equation were computed below :vm (g plant−1 °C−1 d−1 for plant biomass cumulation) is the maximal slope and is deemed as the maximal growing velocity in the fast-growing stage; tm (°C d) denotes the relevant time of vm and denotes the time when growing velocity registers its maximal value; t1 and t2 (°C d) denote the inflexion of the logistic equation and represent the nodal points of the start and end time of the fast-growing period, respectively; and td denotes the duration of the fast-growing period (°C d).where Accumulated thermal unit is adopted in the experiment farm with a small meteorological station, rather than calendar days posterior to cultivation. The formula adopted to calculate thermal time stands thus:Tmax and Tmin denote the daily maximal and minimal temperature (°C), separately; and Tbase denotes the basic temperature of 10°C for greenhouse tomato  before and after SAL treatment is shown in The parameters for fruit quality  were significantly enhanced with increasing SAL . The valF-values between plant growth indicators  and yield showed very significant correlations and indicated that regression analysis could be performed , followed by water consumption (35.7%) and biomass (32.4%), with the lowest value for plant height (4.0%) . The F-verformed .Path analysis showed that biomass had the strongest effect on yield, mainly based on the direct (1.523) and indirect effects of water consumption (−0.609) . Water cvia logistic equations  range was 0.09–0.82 with an average of 0.56, and coefficients of determination (R2) were 0.977–0.994 with an average of 0.987.The path analysis showed the significance of biomass on yield. As the logistic equation performed well in the kinetic description of plant growth with the parameters, it was fitted to the association between accumulated temperature and kinetic variations in tomato biomass. Biomass for diverse SAL and FT treatments was fitted quations . The root1, t2, and tm rose with increasing SAL (t1) for S3.0 and S4.5 was 6 and 12 days later than for S0.8, respectively, with a slope of 3.2 days (dS m−1)−1 (R2 = 0.988). The end of the rapid growth period (t2) of S3.0 and S4.5 was 4 and 28 days later than S0.8, respectively, with a slope of 6.39 days (dS m−1)−1 (R2 = 0.784). The periods of most rapid growth (tm) of S3.0 and S4.5 were 10 and 20 days later than S0.8, respectively, with a slope of 5.06 days (dS m−1)−1 (R2 = 0.981). The maximal growing velocity in the fast-growing stage (vm) dropped with increasing SAL.The values of sing SAL . The sta−1). The decrease of leaf chlorophyll content was associated with salt-caused increase in chlorophyllase activity, which casted an unfavorable effect on membranous stability and attenuation of protein–colorant–lipin complex  of SAL stress and FT remarkably reduced the biomass, dry matter, and chlorophyll, no difference was evident with varied FT conditions. This indicated that tomato growth variables were reduced due to SAL stress but not FT.−1 and above, with a 7.2% yield decrease per unit rise in SAL. Moreover, Magan et al.  were slightly higher than CN condition. This result indicated that the FT method (3FT) in this study failed to achieve the threshold for marketable yield. We may attempt in subsequent experiments to retain four or more fruits to maximize marketable yield. This result also confirms the different effects of SAL stress and FT on tomato yield and marketable yield. Salinity stress reduces yield and marketable yield, whereas reasonable FT can increase yield and marketable yield by increasing weight per fruit. The values of yield variables also exhibited slight differences under the same SAL condition . This result further elucidates that the loss of tomato yield due to SAL stress can be compensated by reducing fruit load and removing substandard fruit in advance, thus achieving no reduction in yield or even an increase in marketable yield. In other words, proper FT can alleviate the effect of SAL stress on tomato yield.via the osmosis effect and afterward induces water stress. Ahanger et al.  between the fruits and plant growth .The experimental results showed that tomato growth variables were significantly decreased with increasing SAL, but FT treatment had less effect on tomato growth.Salinity stress was confirmed to improve fruit quality and reduce fruit cracking, but reduced marketable yield by reducing fruit size. FT was confirmed to improve fruit quality and marketable yield by removing unhealthy fruit and increasing remaining fruit size, but increasing fruit cracking. Combined treatment of SAL stress and FT avoided the problem of subsize fruit under SAL stress treatment, reduced the fruit cracking found with FT treatment, and greatly improved marketable yield and fruit quality.−3, 0.86 × 10−3, and 0.87 × 10−3 mm plant−1, respectively. The daily water consumption of tomatoes was significantly lower for soilless than for soil cultivation. The SAL thresholds of WUE variables were determined to be approximately 3.0 dS m−1 under soilless conditions using Vegetable Life A nutrient solution.Salinity stress suppressed water absorption of tomato, but FT had no effect on water consumption. The daily water consumption before SAL treatment, after treatment, and for the whole growth period was 0.92 × 10t1), end of the rapid growth period (t2), and most rapid growth time (tm) and SAL stress showed that increasing SAL by 1 dS m−1 resulted in 3.20, 6.39, and 5.06 days of delay, respectively. FT tended to advance and shorten the period of plant growth.Path analysis revealed that biomass and water consumption were vital indices positively influencing yield. Based on the logistic equation fitting, the SAL stress tended to inhibit and delay plant growth. The rapid growth period , Education Department of Shanxi Province (2019L0781 and 2021L436), and Shanxi Federation of Social Sciences (2019B375 and 2020YY207).SJ was employed by Eurofins Technology Service (Suzhou) Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The aim of this study was to determine the most efficient protocol for cryopreservation of ovarian tissue using the automatic Freeze Control® system and to test two different cooling curves combined with two different cryoprotectants: dimethyl sulfoxide (DMSO) and ethylene glycol (EG).P<0.05 defined as significant.In this study, 20 female Wistar rats underwent bilateral oophorectomy. The ovaries were divided into two groups: one cryopreserved in 1.5M DMSO and the other in 1.5M EG. Two cooling curves, slow (1h 50min) and rapid (35min) were analyzed. Tissue samples were frozen, thawed, fixed, and stained with hematoxylin and eosin to analyze oocyte integrity. Follicular analysis was performed under optical microscopy (400x magnification) and preantral follicles were classified as primordial or primary according to developmental stage. ANOVA was performed, and Tukey’s test was used for comparison between means, with In cryopreserved tissue, the follicles with preserved integrity in each ovary were 79% primordial and 29% primary. In non-frozen (control) tissue, all follicular types were observed . Reversible changes included cytoplasmic vacuolization and irregular cell outline. Irreversible changes included pyknosis. EG was more efficient than DMSO, preserving a greater number of viable primordial and primary follicles. Comparison of both cooling curves revealed no statistically significant differences between them.The EG is more effective as a cryoprotectant than DMSO for obtaining higher viable numbers of primordial and primary follicles from rat ovarian tissue. Further studies are needed to demonstrate ovarian functionality, such as detection of hormone levels. The survival rates of patients undergoing cancer treatment have been rising in recent years . In an aThe study sample comprised 20 female Wistar rats, approximately 8-12 weeks old. The rat animal model was selected due to the gross similarity between the reproductive systems of human beings and other vertebrates . In the 2 inhalation.Although the rat is a polytocous rodent, the female has a rapid regular ovarian cyclicity of 4 to 5 days, making it a good model for studying histologic changes during cryopreservation. Animals were kept on a 12-hour light/dark cycle with a controlled temperature of 22±2°C and were provided food (standard rat chow) and water ad libitum, in accordance with Brazilian National Health Council Resolution 196/96. Bilateral oophorectomy was performed immediately after euthanizing by COAll procedures were performed in accordance with the Guide for the care and use of laboratory animals , in accordance with Biosafety Brazilian Law N° 11.794, 2008 and with prior approval of the local Animal Research Ethics Committee (CEUA/HCPA) under the number GPPG 10-0255.After bilateral oophorectomy, ovaries were bisected, rinsed with PBS to remove excess blood, and placed in human tubal fluid (HTF) embryo culture medium for 2 min. Next, part of the material was placed in 400 µL of 1.5 M DMSO in HTF and the remaining material was placed in in 400 µL of 1,5 M EG in HTF for 15 min at room temperature. Control group ovaries were merely rinsed with PBS to remove excess blood and placed in 10% buffered formalin.After incubation with DMSO or EG in cryovials, tissue fragments were placed in a Freeze Control® automatic, controlled-rate cryopreservation system, alternately set to one of two cooling curves: curve 1, a slow curve (1 h 50 min), and curve 2, a rapid curve (35 min). Both curves were set to a temperature range of 20°C to -45°C.Each fresh ovary was subdivided into four fragments. Each sample was submitted to one of the cryoprotectants (DMSO or EG) and cryopreserved under two different freezing conditions, denominated as curve 1 or curve 2. Frozen samples were then placed directly in liquid nitrogen at -196°C.Specimens were thawed 60 days after freezing. After removal from the liquid nitrogen bath, specimens were kept at room temperature for 2 min, placed in a 37°C ice water bath for 2 min to melt, and then rinsed with HTF to remove the freezing solution. Immediately afterwards, specimens were fixed in 10% buffered formalin at room temperature for over 6 h before paraffin embedding.Histological analyses were performed with hematoxylin & eosin-stained, 4-µm-sliced, paraffin-embedded samples. Follicles were assessed by light microscopy (400x magnification) and classified according to the modified In this classification scheme, preantral follicles are classified as follows according to developmental stage: primordial, when exhibiting one layer of sparse, rectangular (flat) pre-granulosa cells surrounding the oocyte; or primary, when exhibiting at least one layer of cuboid or columnar pre-granulosa cells, until the oocyte is encapsulated by a single layer of cuboid granulosa cells.Follicles were examined and classified as preserved or not preserved.Follicles with cytoplasmic vacuolization or an irregular cell outline were still classified as preserved, as these changes are considered reversible.Conversely, follicles exhibiting pyknosis were considered damaged. To evaluate ovarian functionality, primordial and primary follicles were counted in the total area of each ovarian slice.P<0.05) for all analyses.The effects of cryopreservation on follicular integrity were assessed by ANOVA. Tukey’s test was used for comparison between means. The significance level was set at 5% (In hematoxylin & eosin-stained sections corresponding to the control group (non-frozen tissue), all follicle types were present , whereas only primordial and primary follicles were present in cryopreserved sections .The most frequent morphological changes in cryopreserved tissue were cytoplasmic vacuolization and irregular cell outline .In cryopreserved tissue, the follicles with preserved integrity in each ovary were 79% primordial and 29% primary. Microscopy analysis of cryopreserved ovarian tissue after thawing showed that use of EG as a freezing solution leads to a greater number of viable primordial and primary folicles on recovery compared to DMSO (P<0.05).P>0.05) (Comparison of tissues exposed to the two different cooling curves (slow versus rapid freezing) revealed no statistically significant differences in the number of viable primordial and primary follicles (P>0.05) .Several studies have focused on improving techniques for cryopreservation of reproductive tissue, with particular emphasis on preserving the integrity of ovarian tissue. The purpose of the present study was to assess the effects of freezing solutions with two different cryoprotectants (EG and DMSO) combined with two different cooling curves, one rapid and one slow, on an automated cryopreservation system.Efficiency in ovarian tissue cryopreservation was defined as the number of preserved primordial and primary ovarian follicles, which are potentially capable of embryo generation after in vitro fertilization if adequate conditions are met. In this study, a greater number of viable primordial and primary follicles were recoverable from tissue preserved with the EG-based freezing solution, regardless of which cooling curve was used during cryopreservation. This result was most likely due to the lower embryonic cell toxicity and higher cell membrane permeability of EG .There were no statistically significant differences in the efficacy of the cooling curves used in this study. This result indicates that, for conventional freezing, the conventional freezing process can be shortened without damaging cryopreserved tissue.Several authors have proposed slow cooling techniques, as ovarian tissue is highly tolerant to the cryopreservation process (The authors believe this may be due to contamination of liquid nitrogen, which is used in freezing and storage and is susceptible to microbial contamination (Overall, a good cell freezing protocol is one that ensures the stability of cross-reactions between molecules in the intracellular environment and enables cell survival after thawing. Viable tissues should not only permit cell survival but also exhibit similar baseline architectures, including cell junctions, interactions, and molecular bonds . CryobioIn this study, regardless of the cooling curve, EG was superior to DMSO as a cryoprotectant for cryopreservation of ovarian tissue in the Wistar rat, as it was associated with a greater number of potentially viable primordial and primary follicles on histological analysis. As there was no significant difference between cooling curves, after histological evaluation one may conclude that conventional rapid freezing protocols produced a superior number of viable primordial and primary follicles obtained from rat cryopreserved ovary tissues.


India is one of the oldest maritime nations in the world, and the overseas contacts date back to the third millennium BCE. Besides several archaeological vestiges, numerous stone anchors of various types have been documented during maritime archaeological explorations along the Indian littoral. During a recent maritime archaeological exploration, a broken Indo-Arabian stone anchor, of the Medieval period, was discovered along the Manikapatna coast of Odisha, Indian eastern littoral. In an attempt to determine the provenance of the anchor, we carried out a detailed petrographic, geochemical (major/trace elements) and Sr–Nd isotopic investigation. The results of our study reveal that the stone of the anchor had been cut out of a geologically young, vesicular, subalkalic basalt lava flow. Source fingerprinting done using petrographic, geochemical and isotopic data, suggests that contrary to the general perception, the anchor rock did not come from any local rock formations. All data point to the most likely scenario that the anchor rock was sourced from one of the lava flows of the Deccan Traps at Palitana in the Saurashtra region of Gujarat, western India. This result confirms the existence of Medieval maritime trading between western and eastern Indian states. A wide range of stone anchors, particularly composite, Indo-Arabian, ring stone and single hole types, have been recorded during maritime archaeological explorations along the Indian coast5 , Rajaraja Chola (985–1016 CE), Sahassamalla of the Polonnaruva period Sri Lanka and Chinese (14th century CE) as well as Chinese Porcelain (1368–1644 CE) belonging to the Yuan and Ming dynasties9.The Odisha Institute of Maritime and Southeast Asian Studies (OIMSEAS), Bhubaneswar carried out the excavations at Manikapatna between 1989 and 1993amalaka (notched stone disk), perforated window pieces, pillar segments, dressed stone blocks. During the digging of the foundation for the construction of a new mausoleum these remains were recovered, which apparently belonged to a temple .During the 2018–2019 exploration adjacent to the mosque of Manikapatna, a broken Indo-Arabian Fig.  stone anple Fig. A. In addple Fig. B of the The Indo-Arabian stone anchor of Manikapatna was broken when discovered Fig. A. Howeve13 (1977). Multiple international rock standards were used for calibration and the BHVO-2 international basalt standard was used for accuracy check.A couple of thin sections of the anchor rock were prepared and petrography was done using a polarizing microscope. Minerals in the rock were identified using their optical properties Fig. A. Major 3 acid mixture and the solution was diluted in 2% HNO3. Measurements were done on Thermo Fisher make Element XR HR-ICPMS and instrumental drift was corrected using internal standards of Ga, In and Bi. Reproducibility was better than 1% (2σ) for rare earth elements, and 2% (2σ) for other trace elements based on repeated analyses of the BHVO-2. For Sr and Nd isotopic ratio analyses the sample powder was dissolved using the standard HF-HNO3-HCl dissolution protocol for silicates. Pure Sr and Nd were extracted by means of conventional cation exchange liquid chromatography with AG® 50W-X8 resin from Biorad and Ln-spec resin from Eichrom®, respectively, and using dilute HCl as elutant. 87Sr/86Sr and 143Nd/144Nd were measured on a Thermo Fisher make Triton Plus TIMS in static mode. Isotopic ratios were corrected for machine induced mass fractionation using internal constant ratios of 0.1194 and 0.7219, respectively for 86Sr/88Sr and 146Nd/144Nd and an exponential fractionation law. The measured values of 87Sr/86Sr of NBS 987 and 143Nd/144Nd of JNdi-1 were 0.710250 ± 0.000008  and 0.512104 ± 0.000004 , respectively.For elemental analyses, the powdered sample was digested in HF-HNO143Nd/144Nd) is also presented as εNd(0) in Table Nd(0) = [(143Nd/144Nd)sample/(143Nd/144Nd)chondrite − 1] × 104, where (143Nd/144Nd)chondrite is taken as 0.512638. The εNd(t) in Table The results of elemental content and isotopic ratio analyses of the anchor rock sample are presented in Table 2O3 and 50.6 wt% SiO2 which are characteristic of a basalt. In the Total Alkali Silica (TAS) classification of volcanic rocks, the anchor rock plots well within the field defined for basalts  = − 12.1) and 143Nd/144Ndi = 0.511956 (εNd(t) = − 11.7), respectively.The rock has 9.51 wt% of MgO, 12.99 wt% Al22  is much higher, and εNd (− 12.1) is much lower than those expected for an uncontaminated young (< 120 Ma) mantle derived basaltic magma. These data hint at presence of significant amount of radiogenic Sr and non-radiogenic Nd in the parental melt that crystallized to produce the source rock of the anchor. Such chemical affinity is usually attributed to continental crustal material, which in turn suggests that the parental magma was contaminated by the crust through which it had erupted.The Manikapatna anchor rock is a basalt, as inferred from its mineralogy, texture and major element chemistry Fig. . It has 22 Fig. A. The me24. Besides, Indo-Arabian stone anchors have also been found during an inland survey of Aramda, Gopnath, Hatab, Ghogha and Mithi Virdi off the Gujarat coast25. Maritime explorations off Tamil Nadu26, east coast of India and Maharashtra29, Goa30, Kerala31, and Lakshadweep32 have also discovered Indo-Arabian stone anchors. More of these Indo-Arabian type stone anchors have been retrieved from the west coast of India compared to the east coast. A large number of these anchors, including the one that is the subject of the current study, have been found in localities adjacent to mosques, which possibly attest to their links to Arab (Islamic) mariners.The Indo-Arabian and other types of stone anchors have been recorded from maritime archaeological explorations in Dwarka, Bet Dwarka, Somnath, Miyani and Visawada of Gujarat, western India33. However, considering that similar rock formations are found elsewhere in India and in Arabian/Iranian territories, which had maritime contact with ancient India, it is imperative that more robust fingerprinting techniques such as (isotope) geochemistry is used for the purpose.The determination of provenance of the anchor rocks is closely linked to the understanding of the trading routes in the ancient world. Since the typology of the anchors and the compositions of the rocks are so diverse that it is not yet fully deciphered whether most of them originated from local rock formations. Besides, very few studies had employed two of the most effective tools, the geochemical and isotopic methods, to address this issue. Earlier studies on Indo-Arabian anchors discovered in India have suggested their origin primarily from Gujarat region34. However, a simple comparison of the measured Sr and Nd isotopic compositions of the anchor rock with those of the basalts of the Arabian region indicates that the anchor definitely did not come to these volcanic rock formations  vs. Zr/Y plot  mentioned Manikapatna as a port where salt taxes were collected38. Bowery (1669–1679) mentioned the maritime activities of Manikapatna port, which was located along the coast of Gingelly39 and engaged in the transportation of food grains and calicoes40. Similarly, a Persian inscription41 found at the mosque of Manikapatna states that Mohammed Kamal constructed the mosque at Manikapatna in 1193 Hizra (1779 CE), during the time of Shah Alam II, the Mughal Emperor. Among several Muslim pir tombs of Odisha, Manikapatna42 has one. It could be assumed that either an Arab or Muslim mariner might have brought the stone anchor to Manikapatna and kept it at the mosque of Manikapatna as a symbol of passion because such examples have been reported earlier from both the east and west coasts of India. The exact reason behind the abandonment of the anchor remains unknown.The chemical and isotopic fingerprinting of the stone anchor of Manikapatna ascertains that the provenance of the rock was Palitana in the Bhavnagar district Saurashtra region of Gujarat Fig. . A marin43. This trade finds mentioned in “Ain-i-Akbari” or the "Administration of Akbar". The traders of the Cambay region primarily dealt with the textiles of Odisha44. The cotton textiles of Odisha, Bengal, Gujarat and Tamil areas were also supplied to the Southeast Asian markets since at least the fifteenth century45. Because such contacts existed, the Indo-Arabian stone anchor of Manikapatna could likely have been brought by some traders of the Ghogha region of Gujarat. Since the distance between Ghogha  and for more than a century afterwards556–1605 The chemical and isotopic compositions of the stone anchor of Manikapatna on the Chilika Lake, Odisha, closely resemble that of the subalkalic basalt lava flows of the DVP near Palitana in the Bhavnagar district Saurashtra region of Gujarat. The absence of such young basalt in Odisha suggests that the stone anchor was transported from the Saurashtra coast. The provenance study of the stone anchor proves direct maritime contacts between Odisha and Gujarat during the Medieval Period, and this could be further corroborated with the references made in the historical records. Further explorations along the Chilika coast may yield more such stone anchors, which would provide a further understanding of the regional and external interactions of India during ancient times.Despite the advancement of tools and technology in the field of shipping, and shipbuilding, i.e., wood to steel and sail to steam and stellar to satellite navigation, traditional fishermen preferred the Indo-Arabian stone anchors until very recently, suggesting continuity of ancient traditions/usages. Thus, it is essential to study and understand the indigenous knowledge of shipping and maritime Archaeology.