Psyllium husk powder was investigated for its ability to improve the quality and shelf life of gluten-free bread. Gluten-free bread formulations containing 2.86%, 7.14%, and 17.14% psyllium by flour weight basis were compared to the control gluten-free bread and wheat bread in terms of performance. The effect of time on crumb moisture and firmness, microbial safety, and sensory acceptability using a 10-cm scale was assessed at 0, 24, 48, and 72 h postproduction. Crumb firming was observed during the storage time, especially for the control gluten-free bread, which had a crumb firmness 8-fold higher than that of the wheat bread. Psyllium addition decreased the crumb firmness values by 65–75% compared to those of the control gluten-free bread during 72 h of storage. The longest delay in bread staling was observed with a 17.14% psyllium addition. The psyllium-enriched gluten-free bread was well accepted during 72 h of storage, and the acceptability scores for aroma, texture, and flavor ranged from 6.8 to 8.3, which resembled those of wheat bread. The results showed that the addition of 17.14% psyllium to the formulation improved the structure, appearance, texture, and acceptability of gluten-free bread and delayed bread staling, resembling physical and sensory properties of wheat bread samples during 72 h of storage. Therefore, according to the obtained results, this approach seems to be promising to overcome some of the limitations of gluten-free breadmaking. The key role of wheat gluten in breadmaking and bread quality cannot be replaced by a single ingredient. Hence, gluten-free (GF) breadmaking basically removes the most crucial ingredient for bread structure and quality. The lack of gluten has a high influence on dough properties, the breadmaking process, and the final quality and shelf life of gluten-free bread (GFB) ,2. As a There has been a rise in the number of people adhering to the GF diet partly due to an increased prevalence and awareness of gluten-related disorders, especially celiac disease, which has become a notorious public health problem worldwide but mainly due to the widespread belief that a GF diet is healthier and more suitable for weight management ,4,5. ThuHowever, GFBs are characterized by a shorter shelf life than wheat bread, owing not only to starch retrogradation and the migration of water from the crumb to the crust but also  to the starchy raw materials and high water content used, two factors that accelerate staling and increase susceptibility to microbially driven deterioration . Thus, cRecent literature reviews, such as those of Cappelli et al. 2020)  and Capr020  and Plantago ovata seeds, can improve health, aiding in intestinal transit, cholesterol control, satiety [Therefore, the use of soluble dietary fibers has been suggested to improve the physical properties, sensory acceptance, shelf life, nutritional content, and glycemic response of GFB ,15. Amon satiety , and gly satiety .In addition to the health benefits, psyllium (P) presents water-binding, gelling, and structure-building properties that can increase the dough viscosity, strengthen the boundaries of the expanding cells, increase gas retention during baking, and improve the volume, in addition to reducing the loss of crumb moisture, softness, cohesiveness, and springiness during storage, thus improving the structure, texture, appearance, acceptance, and shelf life of the GFB ,19,20,21Among the studies cited, the one by Fratelli et al. (2018)  was the Based on previous findings, the present study aimed to investigate the ability of P to improve the quality and shelf life of GFB. Physicochemical, microbiological, and sensory analyses were performed to compare the P-added GFB formulations to the control GFB and wheat bread (WB) during 72 h of storage.® Psyllium P95) was supplied by JRS Latinoamericana Ltd. . This is a dietary fiber concentrate from milled psyllium husks with 95% purity, an average particle size of 250 µm, and an approximately 80% dietary fiber content, as previously described by Fratelli et al. (2018) [The psyllium husk powder (VITACEL. (2018) . The othThe ingredients purchased in the local market  used in the preparation of GFBs consisted of rice flour , cassava starch , calcium propionate , bread spray mold inhibitors , and water, eggs, sugar, soybean oil, salt, and dry yeast.The WBs were prepared with commercial wheat flour , which had contents of ash 0.5%, fat 1.6%, protein 12.3%, total dietary fiber 3.2%, and available carbohydrates 82.4%. The wheat flour had 23% wet gluten according to the manufacturer’s instructions.The GFBs were selected based on a previous study by Fratelli et al. (2018)  and wereThe GFBs were prepared according to the procedure described by Fratelli et al. (2018) . BrieflyTwo WB formulations were prepared as described by Santos et al. (2018) . The firThe WBs were prepared as previously described by Santos et al. (2018) . WB1 wasThe GFBs and WBs were then sprayed with mold inhibitors, packed in polypropylene bags, and stored in controlled conditions  for 3 days. The bread samples were analyzed 0, 24, 48, and 72 h postproduction. Four random loaves were used to monitor instrumental parameters and microbial growth at each storage time. In this way, a total of sixteen loaves were prepared for each bread type.After the microbiological safety was confirmed, twenty-four loaves of each formulation were produced for the sensory evaluation. Six loaves were used to monitor the sensory acceptability at each storage time for each bread type.Fresh bread characterization consisted of the loaf-specific volume, crumb moisture, crumb firmness, crumb grain, and height/width ratio of the central slice, and sensory acceptability. The loaf-specific volume was determined according to AACC method 10–05.0 . For the2) and converted into a grayscale image. The image was binarized to allow the conversion into black and white. Black dots represent alveoli. An alveolar threshold of 0.0005 mm2 was applied. After this processing, the number of cells, the average cell size, and the percentage of cell area were determined, and the crumb grain characteristics were considered. Three slices were analyzed per treatment.The crumb cell structure of the digital bread slice images was assessed by adapting the method proposed by Santos et al. 2021 . The sliSalmonella sp./25 g, according to bread microbiological standards established by Brazilian food legislation [Salmonella spp. was performed from selective enrichment [The effects of ageing on crumb moisture and firmness were monitored in accordance with . To monirichment , and therichment .With respect to the panel test, all subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of the Federal University of Sao Paulo CAAE: 44457415.5.0000.5505.The sensory acceptability of each fresh and stored bread type was evaluated by 54 untrained panelists in one session. Bread consumers were recruited from the campus via internal announcements. They had no gluten-related diseases, had a habit of consuming bread, did not have any allergy or intolerance to any of the ingredients present in the products, and were aware that they were tasting GFBs and WBs. The panelists scored the aroma, texture, and flavor of the formulations on a 10 cm hybrid hedonic scale . SamplesDifferences in the treatment means were identified using analysis of variance (ANOVA one-way) and Tukey’s test. A simple linear correlation (Pearson correlation coefficient) was also evaluated. These data were processed using Statistica 12.0 statistical software . Multiple factor analysis (MFA) and regression vector (RV) coefficients were applied to verify the data relationship using six tables of variables . In the sequence, hierarchical clustering analysis (HCA) and a dendrogram chart were performed based on coordinates of the matrix of factors obtained by MFA, considering Euclidean distances, Ward’s method, automatic truncation, and the cophenetic correlation coefficient, using XLSTAT 2021.1 software .The control GFB (GFB0P) presented half the specific volume and a 9-fold higher crumb firmness than WB1 and WB2. The P addition levels with proper water levels increased the specific volume of GFB by approximately 50% and decreased the crumb firmness by approximately 70% compared to the control. The P-added GFBs  had three-quarters of the volume and a 2.2- to 3.1-fold higher crumb firmness than the WBs.GFB0P presented the lowest loaf-specific volume in a rectangular format (height/width ratio of 0.7) and a compact structure characterized by crumbs with a few large holes because of the cell coalescence and gas escape during breadmaking due to the more liquid batter consistency. The P-added GFB had an improved crumb grain with 2.2- to 2.7-fold more cell area than GFB0P, as shown in The P-added GFB presented a higher loaf-specific volume and lower crumb firmness than the control GFB. WB1 and WB2 presented higher volumes and lower crumb firmness values than the GFB samples developed in this study. WBs presented a lower crumb moisture due to the lower water levels in their formulations compared to those of the GFBs.During bread storage, physicochemical and microbial alterations can affect the sensory parameters of bread, especially the aroma, texture, and flavor . TherefoFresh P-added GFBs did not significantly differ in their physical properties and acceptability scores because the proper combinations of P and W levels defined by Fratelli et al. (2018)  were appThe results show that increasing the percentage of cell area and the loaf-specific volume decreased the crumb firmness, which improved the texture acceptability scores, and the following correlations were obtained between the loaf volume and crumb firmness , the loaf volume and texture acceptability , and the crumb firmness and texture acceptability . The following correlations were obtained between the percentage of cell area and loaf volume , crumb firmness , and texture acceptability . These data show the importance of instrumental indices of bread quality for GFB texture acceptability. In addition, a corroboration between the instrumental and sensory parameters considering the product elaborated in the present study is shown.Crumb firming  was observed during storage, as shown in GFB0P exhibited firmest crumbs 0, 24, 48, and 72 h post-baking b. The adDespite the higher P levels used in the present study, as well as the differences in formulations and process conditions, the results agreed with those of Cappa, Lucisano, and Mariotti (2013) , who repIn view of the staling behavior, GFB7.14P presented no advantage over the GFB2.86P formulation. Thus, considering a good cost-to-benefit ratio, GFB7.14P was not included in the investigation of the time effect on the sensory acceptability of GFB.Salmonella sp. were absent, and no molds or yeasts were detected in the breads during storage.The results of the microbiological safety evaluation indicated that the formulations GFB0P, GFB2.86P, GFB17.14P, WB1, and WB2 are suitable for human consumption based on the current legislation. The microbiological standards of Brazilian food legislation were maintained ; thermotNo significant difference in the aroma acceptability scores was observed for the GFBs during storage. However, a decrease in aroma acceptability was observed for WB1 after 72 h and for WB2 after 48 h. During the 24–72 h of storage, GFB2.86P presented the lowest aroma score, and no difference was observed among GFB0P, GFB17.14P, WB1, and WB2 during storage, which all maintained acceptable aroma scores (scores ranging from 7.1 to 7.8 when measured 72 h after baking).GFB0P and GFB2.86P suffered a significant decrease in texture and flavor acceptability during storage. After 24 h of storage, the texture scores ranged from 4.5 to 5.7, and flavor maintained a lower score , indicating that the developed breads were not acceptable compared with the other breads during the same period. GFB17.14P suffered a significant decrease in texture and flavor acceptability after 72 h of storage, WB1 suffered a significant decrease in texture and flavor acceptability after 72 h and 48 h of storage, respectively, while for WB2, a difference in texture and flavor acceptability was found after 48 h and 72 h, respectively. GFB17.14P was well accepted during the 72 h of storage, with acceptable scores for aroma, texture, and flavor ranging from 6.8 to 8.3, which were comparable to the scores of WB1 and WB2.The panelist scores agreed with the texture instrumental analysis, and a good correlation was found and showed that increasing crumb firmness during storage decreased the bread texture acceptability scores . These data show the importance of these instrumental indicators for bread quality, and crumb firmness would be a predictor of texture acceptability of both fresh and stored WB and GFB. The results show that the addition of 17.14% P to a GFB formulation successfully improves the structure, appearance, texture and acceptability, and delays bread staling. This P-enriched GFB can be considered high in fiber, since it contains 9.2% dietary fiber, and it also has a low glycemic index (GI = 50) and low glycemic load (GL = 9), as previously reported ,35,36.It is important to highlight that the volunteers had no dietary restrictions, and a good acceptability evaluation by this group implies a similarity with regular products. This is very promising for consumers with gluten-related disorders because gluten-free products are often less tasty and less attractive than regular products .Factor 1 explained most of the variance (71.59%). Factor 1 was positively associated with the number of cells and cell area, as well as all the sensorial properties (except aroma at 48 h) evaluated during storage, compared to samples WB1 and WB2. Negatively, the average cell size referred to sample GFB2.86P, and the moisture and firmness during all evaluated storage periods described the sample GFB0P. Factor 2 (18.05%) discriminated only aroma at 48 h, which distinguished sample GFB17.14P.These data indicate that the consumer scores agreed with the texture instrumental analysis, since increasing the crumb firmness during storage decreased the bread texture acceptability scores, as indicated by the inverse position of the vectors a and theThese findings indicate that the GFB17.14P sample resembled the physical and sensory properties of the WB1 and WB2 samples during 72 h of storage.Therefore, the results show that the addition of 17.14% P in a GFB formulation successfully improves the structure, texture, acceptability, and delays bread staling, similar to traditional bread based on wheat flour.The results showed that psyllium addition improved GFB structure, mouthfeel, and acceptability. No significant differences were observed in the acceptability of fresh psyllium-enriched GFBs and WB. During storage, the control GFB had a crumb firmness eightfold higher than that of WB. Psyllium addition decreased the crumb firming ratio by 65–75%. The longest delay in GFB staling was observed with the addition of 17.14% psyllium, maintaining acceptability during storage comparable to that of the WB counterparts. Therefore, our findings are useful for both GFB researchers and producers, indicating a promising alternative for obtaining healthier GFB, together with a reduction of bread staling.


Acute pancreatitis was induced by biliopancreatic ligation in the control and eugenol groups, but not in the Sham group. In the eugenol group, eugenol was administered per-os. Five histopathological parameters, such as edema, inflammatory infiltration, duct dilatation, hemorrhage and acinar necrosis were evaluated.at 72 h from acute pancreatitis induction, the total histological score was diminished in the eugenol group (p<0.0005) and duct dilatation and inflammatory infiltration were reduced compared to the control group (p<0.05). In addition, at 72 h, eugenol reduced pancreatic myeloperoxidase activity (p<0.0005).eugenol, a highly free radical scavenger agent, may have a preventive role in acute pancreatic injury, as it was evident in our rat experimental model. Acute pancreatitis (AP) accounts nowadays for many incidents, with the large proportion of patients being admitted to the emergency room in a critical condition. Early diagnosis is vital towards the right decision in therapeutic approach, in order to have a good outcome. Despite the fact that clinical research has significantly advanced in recent years, mortality still remains high 10%) [0% 1-4]..4]. The As far as the pathophysiology of AP, the cascade of inflammation is thought to begin by intracellular activation of pancreatic proenzymes, which along with enhanced pancreatic duct permeability initiate pancreatic autodigestion. In addition, anti-inflammatory cytokines are produced . AlthougThe most common complication in AP and severe acute pancreatitis (SAP) is the acute lung injury that leads to ARDS followed by renal injury . The mosEugenol  is a food additive or/and drug ,14, whicThe aim of this study is to evaluate the pathologic alterations and adjustments of pancreatic tissue and evaluate the possible diminishment in the degree of acute pancreatic tissue damage after administration of eugenol in a rat experimental model of acute pancreatitis. Furthermore, pancreatic myeloperoxidase (MPO) activity has been thoroughly studied, since it has been shown that it might be used as a marker of neutrophil migration in acute pancreatitis and has been correlated to the severity of pancreatic injury in previously published studies .Experimental animals: we used 120 male Wistar rats . The animals were provided by the Hellenic Pasteur Institute  and were maintained in cages with free access to food and water, under standard laboratory conditions . The experimental surgical procedures took place in the experimental facilities of the ELPEN Pharmaceutical Co., Inc., Experimental-Research Center . The general handling of the animals conformed to national, European (directive 86/609/EEC) and international guidelines on the protection of animals used for experimental and other scientific purposes, under protocol number K/2284.Acute pancreatitis induced experiment model:the animals were randomly assigned into 3 groups, as described below: sham , control  and eugenol . The animals were anaesthetized initially by isoflurane; then, 0.25 mL butorphenol  was injected subcutaneously. The animals were then intubated under direct endotracheal laryngoscopy with a 16 G venous catheter, which was then connected to a rodent ventilator  set at 70 breaths/min and a tidal volume of 3 mL. The proper intubation was confirmed by the observation of the chest expansion and retraction and lung auscultation. Following intubation, anesthesia was maintained by a mixture of 93% O2, 5% CO2 and 2% isoflurane.The animals were subjected to acute pancreatitis as follows : a midliPreparation of eugenol solution: pure eugenol  was purchased and prepared in an oily solution in the chemical laboratory of ELPEN. This was achieved with the admixture of pure eugenol in a corn oil solution at a concentration of 1.5 mg/mL.Histopathological evaluation: the histopathological analysis was carried out at the laboratory of histology and embryology, faculty of medicine, Democritus University of Thrace. Tissue samples were placed in 10% buffered formalin solution, and 4 μm paraffin-embedded sections were stained with hematoxylin/eosin. All specimens were evaluated by a pathologist blinded to the sequence of the biopsy specimens. Slides were evaluated with regard to 5 histopathological parameters, i.e. edema, inflammatory infiltration, duct dilatation, hemorrhage and acinar necrosis [necrosis . The altnecrosis .Immunohistochemistry: tissue sections of 4 μm were mounted on glass slides, dewaxed and rehydrated. The kit, En vision HRP, Mouse/Rabbit detection system (K 5007), DAKO Carpinteria, CA) was used. To inhibit endogenous peroxidase, the specimens were incubated with 3% H2O2 (200 mL H2O and 6 mL H2O2) for 15 min in a dark room. Before the primary antibody was applied, the sections were immersed in 10 mM citrate buffer (pH 6.0), rinsed in tris-buffered saline and subsequently heated in a microwave oven (650-800 W) for three cycles of 5 min. The slides were washed with tris-buffered saline before application of the primary antibody in order to reduce nonspecific binding of antisera. Control slides were used as common negative controls for all antibody staining. Sections were then briefly counterstained with Mayer´s hematoxylin, mounted and examined under a Nikon Eclipse 50i microscope .Scoring was assigned according to the proportion of cells with cytoplasmic staining. Positive expression was determined by the number of stained cells. The average labeling index was assessed according to the proportion of positive cells, after scanning the entire section of the specimen. Sections with greater than 10% stained cells were considered as being positive. The results were graded as negative (0) for <10% of cells stained, low (1) for >10% and <30% cells stained, moderate (2) for >30% and <70% cells stained and of high expression (3) for >70% cells stained.Determination of pancreatic MPO activity: the antibody MPO used was myeloperoxidase , DAKO , that was diluted at 1: 400. The pancreatic tissue was homogenized in 50 mmol/L potassium phosphate buffer (pH 6) with 0.5% hexadecyl-trimethyl ammonium bromide. The homogenates were then centrifuged for ten minutes at 40.000, rpm at 4°C. The resultant supernatant reacted with o-dianisidine dihydrochloride and H2O2 and the absorbance was determined spectrophotometrically at 450 nm (and 540 nm as control wavelength). MPO activity serves as a marker for neutrophil infiltration [ltration .Statistical analysis: data were expressed as mean ± standard deviation (SD) for all inflammatory markers. The ordinal nature of inflammatory markers and the violation of normality in distribution lead us to use non-parametric statistical tests. The comparison of variables among the 3 groups was performed using the Kruskal-Wallis test. Pairwise comparisons were done using the Mann-Whitney test. All tests were applied at each time point . All tests were two-sided, statistical significance was set at p<0.05. All analyses were carried out using the statistical package SPSS version 17.00 .Surgical operation was successful and uneventful on all animals, which then resumed normal diet and bowel function. The animals of the sham group showed normal pancreatic parenchyma; no edema, hemorrhage, inflammatory infiltration, duct dilatation or tissue pancreatic necrosis were observed . AnimalsMore specifically, as far as hemorrhage is concerned, at the time of sacrifice, the control group showed less hemorrhage than the eugenol group (eugenol versus control group at 12 and 48 hour time points). However, at 72 hours the eugenol group showed no difference when compared to the control group. Regarding duct dilatation in animals with experimental AP, the eugenol group showed no statistical significant difference when compared with the control group at 6, 12, 24, 48 hours postoperatively. On the contrary, at 72 hour (p<0.05), the eugenol group showed less duct dilatation than the control group with statistical significance . In relaThe total histopathological score for control and eugenol groups was higher in comparison with the sham group at 72 hours and for the whole sample. Eugenol administration resulted in a lower histopathological score in rats with acute pancreatitis (p<0.0005) .Acute pancreatitis is generally characterized as a condition that exhibits hyperamylasemia, pancreatic interstitial edema, acinar cell injury, leukocyte infiltration and hemorrhage. It occurs because of early activation of proteolytic zymogens within the exocrine pancreas leading to autodigestion of the gland ,21. SubsIt has been described in several AP published studies that high levels in serum concentrations of the anti-inflammatory mediators, such as TNF-α receptors, IL-10 and IL-11 play a significant role during the course of the disease ,21,22. IThis study was undertaken in order to determine whether the administration of eugenol has an advantageous result on the pancreatic inflammation in a rat acute pancreatitis experimental model. The usage of eugenol appeared to ameliorate the severity of acute pancreatitis, as indicated by the milder pancreatic tissue injury. To evaluate the extent of pancreatic injury in this experimental model, we examined histopathological parameters such as edema, inflammatory infiltration, duct dilatation, hemorrhage and acinar necrosis by the use of a semi-quantitative scoring system, as previously mentioned.Interestingly enough, regarding the histopathological assessment, we concluded that the histological score was diminished in the eugenol group experimental model at 72 hours from the inauguration of the process, presenting a statistically significant difference, although the specific figures were not always favorable. As far as hemorrhage in rats is concerned, it seems that eugenol did not have any effect upon it, whereas it reduced the duct dilatation at 72 hours compared to the control group. On the other hand, inflammatory infiltration, as another indicator of pancreatic tissue injury, improved at 72 hours. Additionally, MPO expression has been shown to be less apparent in eugenol group from 48 hours to 72 hours.An experimental model of AP which successfully induces the inflammation of the pancreatic tissue caused by biliary obstruction and leads to multiple organ failure resembling human manifestations has been used before ,24. LigaEugenia caryophyllus and Myristica fragrans. Researchers point out its anti-inflammatory, antioxidant, anticancer activity, anesthetic and muscle relaxant abilities [In previously published work , researcbilities ,26,27. Ebilities .It has been demonstrated that eugenol can be used in treatment of several gram-positive and gram-negative bacteria by inhibiting their action using the agar well diffusion method. Furthermore, it has been shown that the growth of fungal infections like candidiasis may be suppressed by the use of eugenol in in vivo studies . Along wThe extent of the injury in pancreatic tissue during AP is well correlated with the levels of free radicals generation. Anti-inflammatory activity of eugenol, by inhibiting PGE2, COX2, IL1b, TNF-α and leukocyte migration, is well documented along with the anti-oxidant capacity of eugenol. The latter feature could be explained by the formation of complexes with reduced metals and in concordance with the anti-inflammatory activity, which is profound at low concentrations. Taking into consideration eugenol´s aforementioned potentials, this might shed light on the observed improvement in AP -33.et al. [Efforts have already been made in experimental animal models, in order to investigate the potential pharmaceutical or phytoceutical properties of eugenol to improve AP. Sowjanya et al.  have shoOur attempt focused on demonstrating the advantageous effect that eugenol brings about in AP. Eugenol, a highly free radical scavenger agent, is the main phenolic compound extracted from certain essential oils. As it was evident in our experimental model, eugenol was found to have preventive role in acute pancreatic injury in rats, which presents a promising finding to continue the study with clinical research in humans.Several studies have shown that eugenol exhibits anti-inflammatory, antioxidant, anticancer activity, anesthetic and muscle relaxant abilities and also exhibits pharmacological effects in almost all human systems;Eugenol seems to have protective role in ameliorating the detrimental effects of L-arginine induced acute pancreatitis in experimental animal models.To our knowledge, the current study is the first evaluating the effect of eugenol in acute pancreatitis experimental model induced by pancreatic duct ligation.


A 68 year-old female presents with an ulcerated mass of the 5th digit, with rapid growth during the previous month to surgery. The mass was excised and covered with a 4th dorsal metacarpal artery perforator flap. The histologic analysis was compatible with the diagnosis of fibro-osseous pseudotumor of the digit. The hand surgeon acts both as an oncological surgeon, ablating all the tumor mass, potentially compromising aesthetics and function, and as a reconstructive surgeon, trying to optimize hand function. Balancing these goals may be a demanding task .Even though the majority of hand tumors are benign, especially if they do not involve the skin, some malignant tumors arise in the hand. In the latter, a more aggressive approach would be justified .Fibro-osseous Pseudotumor of the Digits (FOPD) is a rare benign tumor with clinical characteristics that can mimic a malignant tumor. We present a case of FOPD and a review of the literature.A 68 year-old female, smoker, presented to the ER with an ulcerated mass of the dorsum of the 5th finger involving the proximal phalanx to the DIP joint . It had Excision with the overlying skin was performed down to a tumor less surgical plane, preserving the extensor apparatus . The resFOPD is a rare entity. There are 173 cases documented in the literature , totalinRadiologically, FOPD usually presents as a soft tissue mass with ill-defined margins and might reveal extraosseous ossification and bone formation. The periosteal reaction has been described . CorticaHistologically, FOPD presents as a multi-nodular lesion with irregular margins and it is localized in the dermis and subcutaneous tissues. Muscular and cartilage involvement is absent.Typically, FOPD shows fibroblastic/myofibroblastic proliferation without atypia, in a myxoid stroma, and immature trabeculae with osteoid rimmed by osteoblasts, without zoning phenomenon ,23,51. OImmunohistochemically, the majority of studies has demonstrated positive staining for vimentin 14 ,25,26,34The majority of cases demonstrated negative staining to S – 100, CD 34, cytokeratin (MAK-6 and CAM 5.2), desmin, and epithelial membrane antigen (EMA) ,18,25–27FOPD might be included in a group of USP-6-rearranged myofibroblastic neoplasms that share a genetic rearrangement of the USP-6 gene and are known for their rapid yet self-limited growth and low recurrence rate ,55. ReceThe treatment for FOPD is surgical excision. Prognosis is excellent as recurrence has been related to incomplete excision ,11,42. WNine cases of recurrence were described in the literature ,23,31,43After complete removal of the tumor, no recurrences were found with a median follow up of 15 4–168) months –168 mont,48,53. EFOPD should be considered in the differential diagnosis of fast-growing lesions of the extremities. An excisional biopsy allows for complete tumor removal and avoids overzealous treatment. FOPD should be treated without radical excisions provided if totally removed.


Food legumes are at the crossroads of many societal challenges that involve agriculture, such as climate change and food sustainability and security. In this context, pulses have a crucial role in the development of plant-based diets, as they represent a very good source of nutritional components and improve soil fertility, such as by nitrogen fixation through symbiosis with rhizobia. The main contribution to promotion of food legumes in agroecosystems will come from plant breeding, which is guaranteed by the availability of well-characterized genetic resources. Here, we analyze seeds of 25 American and European common bean purified accessions  for different morphological and compositional quality traits. Significant differences among the accessions and superior genotypes for important nutritional traits are identified, with some lines showing extreme values for more than one trait. Heritability estimates indicate the importance of considering the effects of environmental growth conditions on seed compositional traits. They suggest the need for more phenotypic characterization in different environments over different years to better characterize combined effects of environment and genotype on nutritional trait variations. Finally, adaptation following the introduction and spread of common bean in Europe seems to have affected its nutritional profile. This finding further suggests the relevance of evolutionary studies to guide breeders in the choice of plant genetic resources. Phaseolus vulgaris L.) is the most grown food legume crop worldwide. It is largely grown as a grain crop  and as a fresh vegetable . In 2018, 30.4 million tons of dry seeds and 24.7 million tons of fresh pods were produced worldwide, with a total of 34.5 and 1.6 million hectares destinated for its cultivation, respectively [Common bean . The maP. vulgaris has its origins in Mesoamerica [Genetic improvement is one of the most viable strategies to obtain varieties that in addition to having improved agronomic traits, such as yield and resistance to biotic and abiotic stress, also have the best nutritional characteristics to satisfying consumer needs . Exploitoamerica , from whoamerica ).Phaseolus vulgaris was introduced into Europe in the 16th century, after the discovery of the Americas. It then spread throughout Europe, following complex routes that involved multiple exchanges between different European countries, with continued introduction of new varieties from the Americas [Americas ,19,20,21Seed compositional traits in common bean genetic resources have been evaluated in the literature ,32,33,34In the present study, we aimed to characterize and describe nutritional quality traits of 25 different American and European domesticated genotypes of common bean to identify potential sources of improved seed quality traits that can be used in future breeding programs. We also investigated whether the process of adaptation to European environments had any effects on seed compositional traits.Pv_all, Pv_core1, Pv_Core2) that were developed to be representative of both American and European germplasm, and of the two main gene pools of the species: Mesoamerican and Andean.A total of 25 common bean domesticated accessions were used in this study , 13 fromPv_all, Pv_core1, and Pv_core2 comprise ~10,000, 500, and 200 domesticated lines of P. vulgaris, respectively. The 25 accessions included in the present study were part of Pv_core1. Within the activities planned for the BEAN_ADAPT project, genotyping-by-sequencing of Pv_core1 provided the data for the population structure analysis that highlighted the main structure as two genetic clusters, which represent the Mesoamerican and Andean gene pools .All of the materials are lines of single seed descent that were purified by one or more cycles of selfing under insect-free conditions. This allows association of phenotypes to reliable genotypic information and represents strong potential to promote genetic resources used in pre-breeding and breeding programs. q) >50% used to assign the genotypes to one of the two gene pools, 20 were Mesoamerican, and five were Andean  to the Mesoamerican gene pool, while five Mesoamerican accessions showed introgression from the Andean gene pool . One accession was completely assigned to the Andean gene pool , while the remaining four Andean accessions showed introgression from the Mesoamerican gene pool .In the present study, with a threshold of memberships . This is an internal intensive horticultural area, characterized by a temperate Mediterranean climate with cool winters and warm dry summers. The data of maximum and minimum temperature and rainfall for the growing season (May to October) were obtained from the nearest weather station located, as shown in 2O5) fertilizer was applied as basal fertilization before sowing. Standard conventional agronomic management and irrigation practices were applied to the experimental fields. Plastic cover was used for weed control. Pest and disease management was done by spraying the plants with Ridomil Gold (fungicide) and Klartan 20 Ew (against aphids).The morphology and nutritional quality traits of these 25 accessions were analyzed using seeds produced during a field experiment involving all of the 500 Ten pods for each plot and replicate were harvested at fully maturity  and threshed. Seed weight , cotyledon and embryo weights, and seed coat weight and thickness were determined. For cotyledon, embryo, and seed coat weights, seeds were soaked in distilled water for 24 h. When the seeds were fully hydrated, the cotyledons, seed coat, and embryo were removed and dried in silica gel. When the components of the seeds were completely dry, the weights of the cotyledons, embryos and seed coat were determined using a precision scale, and the thickness of the seed coat was measured with a digital micrometer . Cotyledon, embryo, and seed coat weights for each accession are reported as mg/g seeds.The seeds were lyophilized and milled to a fine powder using a planetary mill with an agate jar and balls . The following seed nutritional traits were measured.Protein content was determined using Dumas combustion. The N content was measured using a N/protein analyzer , as described in the Official Methods of Analysis, Dumas method, 990.03 .v/v) for extraction of the lipid matter, at room temperature and with magnetic stirring. After 3 h, the content was divided in two tubes and centrifuged at 5000× g for 15 min. The lipid-free powder pellet was discarded. The solvent was transferred to a new tube and removed in a rotary evaporator . The remaining oil was recovered with acetonitrile and transferred into a 2 mL vial. The total recovered lipids for each accession was weighed. The lipid content was expressed as g/100 g of dry seed.For the tocopherol analysis, 5 g to 10 g of powdered seeds obtained by lyophilizing and grinding was dissolved in chloroform:methanol , at a flow rate of 0.45 mL/min. Fluorescence detection was at 296 nm excitation and 325 nm emission. For quantification of tocopherols, seven standard stock solutions of γ-tocopherol in acetonitrile were prepared and analyzed .The different tocopherol components were identified according to Giardinieri et al. . BrieflyThe antioxidant activity was measured according to the α, α-diphenyl-β-picrylhydrazyl (DPPH) method . The TroThe percentage of inhibition for the samples and Trolox was calculated according to Equation (1):The TEAC of each sample was as given by Equation (2):Total carotenoid content was reported as yellow pigment content (YPC), according to the micro-method described by Beleggia et al. ,39 and e3 (69.5%)/2 mL H2O2 (30%) using a microwave oven , and then diluted to 50 mL with high purity deionized water acidified with 3% HNO3. The mineral contents were then determined using inductively coupled plasma mass spectrometry  with an auto-sampler , as described by Beleggia et al. [To determine the mineral contents , 20 mg of each sample was dissolved in 8 mL HNOa et al. . BrieflyData for the following 23 traits were used for the analysis : (i) morcorrplot [The statistical analysis was performed using RStudio v1.2.5001 , with thcorrplot .p < 0.05, using the R package agricolae v 1.3-3 [minque v 1.1 [2) was computed as the ratio between genotypic variance and phenotypic variance [Differences among accessions for all of the quality traits considered were tested using one-way analysis of variance (ANOVA). Mean discrimination was performed, by applying Tukey’s tests, and statistically significant differences were determined at the probability level of  v 1.3-3 . To calcue v 1.1  was usedvariance . Wilcoxovariance  was usedFactoMiner v1.42 [factoextra v1.0.5 [ggplot2 v3.2.1 package [The differences between the American and European accessions were tested by an analysis of covariance (ANCOVA), including population structure information as covariate in the model. The multivariate analysis, principal component analysis (PCA), was performed with the packages er v1.42  and facta v1.0.5 . PCA was package .Means (±standard deviation) were computed for all of the traits analyzed in each accession , with thVariability among the different genotypes was seen for all of the traits considered . ANOVA aVariation was also detected for the different parts of the seeds, as cotyledon, embryo and seed coat weights, which ranged from 879.1 mg/g to 924.3 mg/g, from 9.1 mg/g to 16.1 mg/g, and from 62.7 mg/g to 106.2 mg/g, respectively . In partEven though wide ranges were seen for lipid content (1.8–3.1 g/100 g of dry seed), γ-tocopherol (43.3–127.2 mg/100 g of lipids), and the ratio between γ-tocopherol and δ-tocopherol (7.7–19.8), significant differences among the accessions were only seen for δ-tocopherol . Here, tp = 0.001). The carotenoids contents ranged from 2.2 to 9.3 mg β-carotene/kg dry seed  were seed weight, antioxidant activity, carotenoids content, Na, Mo, and Se contents. Intermediate heritabilities (0.30 < h2 < 0.60) were detected for embryo, cotyledon and seed coat weights, δ-tocopherol, protein, and Ca, Cu, Fe, Mn, Ni, and Zn contents. Very low heritability estimates (h2 < 0.30) were seen for the remaining traits, seed coat thickness, lipids, γ-tocopherol, and Mg, P and K contents.Heritability was estimated for all the traits analyzed . The tra2 estimate was for seed weight , followed by embryo weight ; seed coat and cotyledon weights, and seed coat thickness showed low h2 estimates . Among the nutritional quality traits, high h2 estimates were seen for antioxidant activity and carotenoid content . The heritabilities for δ-tocopherol and protein content were 31.5% and 30.5%, respectively. Very low h2 estimates were detected for γ-tocopherol  and lipid content , revealing the strong environmental influences on these traits and/or no differences within these specific materials. Finally, high h2 was seen for Na, Se, Mo, Zn, and Ni . The Ca macro-element and the Mn, Cu and Fe micro-elements showed moderate heritability estimates . Low heritability estimates were seen for the remaining elements (h2 ≤ 22.4%).Considering the morphological seed traits, the highest hANCOVA analysis was carried out to test for significant differences among the American and European accessions for the morphological and compositional traits of the seeds. For the morphological traits, compared to the American accessions, the European accessions showed significantly higher seed weights and lower embryo weights . For theStrong differences were detected for the antioxidant activities and carotenoids content. In particular, these were both higher for the American accessions . The TEAAmerican and European accessions differed significantly for three macro-elements. In particular, compared to those American, the European accessions showed higher Na and Ca contents, and lower Mg content . Within Principal component analysis was performed to investigate the relationships among the accessions, and to determine which seed traits define the basis of their differentiation. Considering PCA analysis based on all of the morphological traits, nutritional traits, and the macro-elements and micro-elements , principHere, PC1 was significantly and positively correlated with seed weight, γ-tocopherol, cotyledon weight, δ-tocopherol, and protein content, and negatively correlated with seed coat weight. embryo weight, YPC, and TEAC , as alsoPC1 tends to separate the American and European accessions, whereby those from Europe have higher mean seed weight and protein content, and higher mean content of both tocopherol congeners, while the American accessions showed higher TEAC and YPC activities , in agreNo clear distinction was detected in these analyses among the accessions, even if the macro-elements and micro-elements also showed wider variability for the European accessions . Europeap < 0.05), and negatively correlated with embryo weight . A strong negative and significant correlation was seen between cotyledon and both seed coat weight and embryo weights . For lipids and γ-tocopherol and δ-tocopherol contents, these were significantly and positively correlated , while γ-tocopherol content was negatively correlated with lipid content  and δ-tocopherol was positively correlated with protein content , but negative correlated with TEAC  and YPC . TEAC and YPC were positively correlated . Significant correlations were also seen between the morphological and nutritional traits; in particular, seed weight was positively correlated with γ-tocopherol content  and protein content , and strongly negatively correlated with TEAC and YPC . TEAC was also positively correlated with embryo weight  and seed coat weight  and negatively correlated with cotyledon weight .Correlations between traits were also investigated . For morp < 0.05). Among the macro-elements and micro-elements, the positive correlations that showed significance (p < 0.001) were those between Ca and Na (r = 0.34), Mn and K (r = 0.43), Mn and Ca (r = 0.41), Mg and Mo (r = 0.34), Mg and K (r = 0.64), and Fe with Ca (r = 0.52), Na (r = 0.42) and Mn (r = 0.33). K and Mg were strongly correlated with the total amount of macro-elements , and Fe content with the total micro-elements content .All of the significant correlations between the different macro-elements and micro-elements were positive, except for the macro-element P, which was negatively correlated with Na, Ca, Mn, and Se , Zn , and Ni . Seed weight was significantly negatively correlated to Mn, Se, and Mo . Significant negative correlation was seen between embryo weight and Na content ; cotyledon weight and Mg content were negatively correlated , while seed coat weight was positively correlated with Mg and Zn .Considering seed weights and macro-elements and micro-elements, significant positive correlations were detected between seed weight and Na  and YPC . YPC and Mn content were strongly correlated .Focusing on the nutritional traits, the macro-elements and micro-elements showed different significant correlations; among the most significant, Na and Zn were negatively correlated with TEAC  the seeds of the different accessions were derived by single seed descent, and are thus identical at the genome level; (ii) the majority of the accessions were landraces, thus potentially including variability that is not present in modern varieties; (iii) the seeds included accessions that were representative of all of the different growth habit types, as a character that is associated with several agronomic traits and with common bean races ,51,52,53Large variability was seen for all the traits analyzed, with the exception of lipid content, for which the heritability estimate was zero, and for seed coat thickness, γ-tocopherol, and Mg, and K contents, for which no significant differences and low heritabilities were seen. Thus, the discussion focuses mainly on the traits that showed significant differences among the accessions and intermediate-to-high heritability estimates.2, 91.4%) in the present study. This was expected, as the literature is full of studies that have reported such observations for common bean [Seed weight was the trait that showed the greatest estimate of heritability . TheIt is well reported in the literature that the antioxidant activity is higher in the seed coat compared to the cotyledons, due to the higher content of phenolic compounds in this part of the seed ,84,85,86The content of total carotenoids (reported as YPC) was also determined here. They are important in human nutrition as a precursor of Vitamin A, as well as antioxidant, with the demonstration that carotenoid-rich diets are associated with decreased incidence of diverse diseases . InteresAntioxidant activity significantly and positively correlated with YPC. A similar result was reported by , who anaThe total tocopherol content was not correlated with either antioxidant activity or YPC, which were instead negatively and significantly correlated with δ-tocopherol. Significant correlation between tocopherols and antioxidant activity has been shown in other species, which suggests that the phenotypic architecture of the antioxidant activity might be highly variable among species. Zhang et al.  reportedAntioxidant activity and carotenoid content were here significantly and negatively correlated to seed weight. This was also reported by Dong-Yun et al.  in an anRecently, there has been an ever-increasing demand by consumers for legumes as sources of protein alternative to meat. This interest arises not only because food legume crops represent valuable resources for diet change, but also because of the relative reduction in environmental impact of such protein production . In thesLegumes are good sources of essential minerals for human health ,95, and Iron is an essential element for human health. It is involved in important metabolic and cellular processes such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis and it is required for synthesis of proteins involved in the oxygen transport, hemoglobin and myoglobin, and other iron-containing enzymes involved in electron transfer . High vZinc is the second most important trace element for human after iron as it is involved in many metabolic processes . It is aHowever, other macro-elements and micro-elements that are important for human nutrition and health are also present in common bean seeds, such as Ca, Cu, Mg, Mn, K, P, and Se . In the Sodium showed the highest heritability, and also significant differences among the accessions. Na is needed for humans, as it is involved in several physiological functions; however, it is well documented that the Na consumption greatly exceeds the minimum daily required intake. Such excess of Na in the human diet can have several negative consequences on health , and thus beneficial effects can be obtained by reducing Na intake . IntereCalcium is a key nutrient for humans, as it is essential for many functions around human health, such as for bone health as it provides rigidity to the skeleton and calcium ions play a role in many metabolic processes. To reach proper Ca intake levels is essential to prevent skeleton system diseases such as osteoporosis and to reduce the risk of bones fractures . Ca intaAmong the micro-elements, very high heritability was seen for Se content, along with wide variability. Of interest here, for 16 of the 25 accessions no Se was detected, while the remaining accessions showed high Se content. Accessions ECa096 (landrace from Costa Rica) and ECe122 (cultivar from Germany) showed the highest Se contents . Four other accessions were characterized by high Se content . Celmeli et al.  reportedThe other micro-elements of Cu, Mn, Mo and Ni showed intermediate to high heritabilities and significant differences among the materials analyzed. The highest contents of each of these four microelements were seen respectively for accessions ECe092, ECa052, ECe234, and ECa095. Determination of the contents of such micro-elements is largely lacking in the literature. The highest Cu content of 16.5 mg/kg and Mn content of 34.8 mg/kg in the present study are similar to the contents reported by Brigide et al.  and PinhSeveral significant correlations were defined here for the nutritional elements. Considering those with significance level < 0.001, the positive correlations that involved the macro-elements and micro-elements were between: Mg and K; Mn and K; Mn and Ca and Fe and Mn ; Mo andThe data obtained in the present study can also be seen from another point of view: i.e., as the evolution of the common bean. One of the main evolutionary processes for common bean was its introduction, spread, and adaptation to European agro-ecosystems.Even if our sample is too small to have a complete picture of the effects of such process in the quality traits of common bean seeds, our findings (both the PCA and ANCOVA) represent a first observation that these events strongly affected not only common bean genetic diversity ,21, but Principal component analysis showed that the European accessions were more widespread compared to the American accessions, which suggests a higher diversity for seed morphology and chemical composition in the European common bean. This major diversity might have arisen in Europe because the frequency of hybrids between the Mesoamerican and Andean gene pool was more than four-fold that in the Americas, which appears to be due to breakdown of geographic barriers and co-cultivation of the Mesoamerican and Andean forms in close sympatry by farmers ,21. The The characterization of plant genetic resources is crucial for breeders, so that they can exploit the diversity to implement appropriate breeding programs. This was the main aim of the present study, where 25 common bean genotypes were analyzed for nutritional traits.One of the main outcomes of the present study is the high variability across these samples for almost all of the traits, and the identification of some very interesting genotypes when considering multiple traits. The Greek landrace ECe092 was characterized by the highest lipid, δ-tocopherol, protein, and Cu contents and very high γ-tocopherol, P, and Ni contents. The Spanish landrace ECe312 showed high γ-tocopherol, δ-tocopherol, protein, K, and Cu contents. The Mexican cultivar ECa095 showed high antioxidant activity and carotenoids, Ca, Mn, and Se contents, the highest content of Ni and low Na content. Focusing on the minerals, three accessions are particularly interesting for future breeding: (i) the Spanish landrace ECe234 showed the highest K and Mo contents and high contents of Mg, P, Fe, and Mn were observed for this accession too; (ii) the German cultivar ECe122 showed the highest Ca and Fe content and was among the highest for Mn, Se, and Mo contents; and (iii) the Italian landrace ECe177 was characterized by the highest P content and particularly high Ca and Mo contents and low Na content.The use of pure lines  and the evaluation of seeds obtained in a designed field trial with replicates allowed estimation of the heritabilities of the different nutritional traits. This parameter is important, inasmuch as it provides information about the breeding value of the sample. Not all of the traits showed intermediate to high heritability estimates, which indicates a relevant influence of environmental conditions for these nutritional compounds. This suggests the need to conduct more studies on the influence of the environment  on the expression of common bean nutritional traits, to identify the best agro-ecological conditions that can maximize the nutritional quality of the promising genotypes.Finally, this study points out the importance to take into account the evolutionary history of crops; indeed, even if more studies are needed in this regard, our findings suggest significant effects on diverse quality traits related to adaptation of the common bean in Europe, out of its centers of origin. Acquiring more knowledge of this evolutionary process can indeed guide geneticists and breeders in their choice of samples to be characterized for different traits.


The fecal microbiota beta diversity of the two calf groups was also significantly different at Wk4 (p < 0.05). Numerous significant differences were present in the fecal microbiota taxonomy of WM and BM calves in terms of relative normalized operational taxonomic unit (OTU) levels, affecting five phyla, seven classes, eight orders, 19 families, and 47 genera. At the end of the trial, when 6 weeks had passed since exposure to WM, the phyla Bacteroidetes, Firmicutes, and Saccharibacteria were lower, while Chlamydiae were higher in WM calves. Notably, WM calves showed a decrease in beneficial taxa such as Faecalibacterium, with a concomitant increase in potential pathogens such as Campylobacter, Pseudomonas, and Chlamydophila spp. In conclusion, feeding pre-weaned calves with unpasteurized WM containing antibiotics is related to a higher incidence of neonatal diarrhea and leads to significant changes in the fecal microbiota composition, further discouraging this practice in spite of its short-term economic advantages.The cows receiving antibiotics for intra-mammary infection (IMI) produce milk that cannot be marketed. This is considered waste milk (WM), and a convenient option for farmers is using it as calf food. However, adding to the risk of selecting resistant bacteria, residual antibiotics might interfere with the gut microbiome development and influence gastrointestinal health. We assessed the longitudinal effect of unpasteurized WM containing residual cefalexin on calf intestinal health and fecal microbiota in an 8-week trial. After 3 days of colostrum, six calves received WM and six calves received bulk tank milk (BM) for 2 weeks. For the following 6 weeks, all 12 calves received milk substitute and starter feed. Every week for the first 2 weeks and every 2 weeks for the remaining 6 weeks, we subjected all calves to clinical examination and collected rectal swabs for investigating the fecal microbiota composition. Most WM calves had diarrhea episodes in the first 2 weeks of the trial (5/6 WM and 1/6 BM), and their body weight was significantly lower than that of BM calves. Based on 16S rRNA gene analysis, WM calves had a lower fecal microbiota alpha diversity than that in BM calves, with the lowest  Waste milk (WM) includes low-quality colostrum, transition or post-colostral milk, milk from cows treated for mastitis and other diseases, milk with high somatic cell count (SCC), and other unsalable milk . AccordiWhen antibiotics are administered to adult individuals with a mature gut microbiome, microbial diversity has been shown to decrease significantly, but resilience mechanisms slowly restore the original condition once antibiotics are removed . On the Given its relevance for the dairy industry, previous studies have assessed the impact of WM on calf health and the gut microbiome , 13, 14,With these premises, we assessed the impact of WM obtained from cows receiving intra-mammary cefalexin on calf intestinal health and on fecal microbiota diversity and taxonomy during 2 weeks of feeding and after up to 6 weeks after the removal of WM from the diet. To reduce variability, colostrum and WM were standardized and characterized before feeding them to calves. The two-step, 8-week trial included 12 dairy calves enrolled in a commercial farm and managed with standard procedures. For the first 2 weeks, six calves received WM, and six received bulk tank milk (BM); for the following 6 weeks, all calves received the same weaning diet with milk whey and starter feed. Every week for the first 2 weeks and then biweekly for the following 6 weeks, we carried out a complete clinical evaluation and collected fecal swabs for investigating the fecal microbiota composition.®, MSD Animal Health S.r.l., Segrate (MI), Italy], and type-1 and type-2 bovine viral diarrhea virus (BVDV) . The farm was followed by our University Hospital Clinic and was selected for its very low prevalence of neonatal calf diarrhea (NCD) in the previous 3 months . The research protocols were reviewed and approved by the Institutional Committee for Animal Care of the University of Milan (protocol number 78_2018). The trial was carried out between March 2019 and June 2019.The study was performed on a commercial dairy farm in Northern Italy with a long-standing collaboration with the University of Milan. The farm included 390 lactating Italian Friesian cows. The herd was accredited free from infectious bovine rhinotracheitis (IBR), vaccinated for neonatal diarrhea agents [Rotavec CoronaThe trial structure is illustrated in ad libitum. In the first 2 weeks, the calves were housed in individual hutches, while in the last 6 weeks, they were kept in two separated collective pens, one for each experimental group. The WM preparation and administration procedures, as well as the composition of WM, BM, and milk replacer, are detailed in section Colostrum, Transition Milk, Waste Milk, and Bulk Tank Milk.Starting from the fourth day of life, six calves were allocated to the BM group and six to the WM group according to the order of birth. For 2 weeks Wk0–Wk2; , BM calvAt birth and on the third day (Wk0), 10th day (Wk1), 17th day (Wk2), 31st day (Wk4), 45th day (Wk6), and 59th day of life (Wk8), all calves were submitted to a complete clinical examination  as detaiTo eliminate possible variables related to colostrum or TM, a pooling strategy was applied as follows. Six liters of good-quality colostrum (Brix >22%) were milked from each of 10 different cows and stored in 500-ml bottles, 12 for each cow. The bottles were identified as colostrum, labeled with the cow number, and frozen at −20°C. Then, 6 L of the second and third milking of the same cows (TM) were again collected in 500-ml bottles, 12 for each cow. The bottles were identified as second-day or third-day TM, respectively, labeled with the cow number, and frozen at −20°C. For colostrum administration, the 3-L morning feeding of each calf was prepared by defrosting and pooling six 500-ml aliquots belonging to cows 1–6, while the 2-L afternoon feeding was prepared by defrosting and pooling four 500-ml aliquots belonging to cows 7–10. The aliquots were gently thawed in a water bath at 45°C for 30 min, mixed, and administered at 35–40°C by oroesophageal tubing. The second-day TM and third-day TM were prepared by mixing aliquots 1–5 for the morning dose  and aliquots 6–10 for the afternoon dose  of the respective TM. In this way, all calves received the same colostrum and TM before the start of the feeding trial.Bacillus spp.; cow B, SCC 901,000 cells/ml, Non-aureus staphylococci (NAS); cow C, SCC 239,000 cells/ml, Staphylococcus aureus; cow D, SCC 5,045,000 cells/ml, Bacillus spp.; cow E, SCC 454,000 cells/ml, NAS.WM was obtained from five cows affected by chronic mastitis (A–E), selected based on a previous bacteriological culture result according to the National Mastitis Council (NMC) guidelines . Ten mic® bags, Dairy Tech Inc.) and stored at −20°C until needed. This collection, mixing, and aliquoting procedure ensured the generation of a uniform pooled WM. WM bags were gently thawed in a water bath at no more than 45°C for 45 min and fed to calves at a temperature ranging from 35 to 40°C. BM was collected fresh from the commercial milk tank.The five cows were subjected to the intramammary administration of 210 mg cefalexin monohydrate  in each quarter for four consecutive milkings, and the milk was collected at each following milking time for a total of 336 L. All the milk was maintained in a refrigerated tank for 36 h from the first to the fourth milking, mixed, aliquoted in 2-L aluminum bags (Perfect Udder® SP NT (DSM). WM was further evaluated in triplicate by liquid chromatography–high-resolution mass spectrometry (LC-HRMS) for antibiotic residue detection and quantitation as described by Chiesa et al.  methods and tested for the presence of inhibitors by the DelvotestEnterococcus faecium DSM 7134 and Lactobacillus rhamnosus DSM 7133 at 1 × 109 CFU/kg. The powder was reconstituted according to the manufacturer instructions (125 g/L of powder).The commercial milk replacer contained milk whey, whey proteins, vegetable oils , hydrolyzed wheat protein, pregelatinized wheat flour, dextrose, butyric acid esters added with vitamins, oligo-elements, and stabilizers of the intestinal flora Clinical examination and calf growth measurements were performed at the six experimental time points Wk0–Wk8;  by an exRectal swabs were thawed, and DNA was extracted using a QIAmp DNA Stool kit  according to the manufacturer instructions with a minor modification. The rectal swabs were dissolved in 1 ml Buffer ASL and shaken at 1,000 rpm  continuously until the stool samples were homogenized. DNA quality and quantity were assessed with a NanoDrop ND-1000 spectrophotometer , and the isolated DNA was stored at −20°C until use.Bacterial DNA was amplified by targeting the V3–V4 hypervariable regions of the 16S rRNA gene . PCR ampDemultiplexed paired-end reads from 16S rRNA-gene sequencing were first checked for quality using FastQC  for an ihttps://github.com/filippob/Rare-OTUs-ACE.git) and R (https://github.com/filippob/sampleBasedRarefaction) scripts. Plots were generated using the ggplot2 R package  and across (beta diversity) samples. All indices  were estimated from the complete OTU table (at the OTU level), filtered for OTUs with more than 10 total counts distributed in at least two samples. Besides the number of observed OTUs directly counted from the OTU table, within-sample microbial richness, diversity, and evenness were estimated using the following indices: Chao1 and ACE for richness; Shannon, Simpson, and Fisher alpha for diversity –38; SimpU-test. Categoric variables were compared with contingency tables and with the Fisher's exact test (2 × 2 tables), calculating the odds ratio. Statistical significance was considered for p < 0.05.The differences between feeding groups were evaluated with SPSS 25.0 (IBM). The distribution of continuous variables was analyzed with the Shapiro–Wilk test. Since the distribution was not normal, data were compared with a non-parametric Mann–Whitney For the microbiome analysis, differences between groups  along time points in terms of OTU abundances and alpha diversity indices were evaluated with a linear model of the following form:p-values were obtained to identify those OTUs and alpha diversity indices that were significantly different between treatments along the six time points of the experiment/trial. Alpha diversity indices: value = mu + group + e, within time point.where y_ij is the abundance (counts) or index value for each taxonomy (OTU) and alpha diversity metric in animal I belonging to treatment group j, treatment_j is either WM or BM, and e_ij are the residuals of the model. From model (1), WM had the following gross composition: SCC 450,000 cells/ml; fat 3.7%; protein 3.6%; lactose 4.7%; microbial inhibitors: present. According to HPLC-MS/MS , WM had During the first 2 weeks of the trial, five out of six (83.33%) WM calves and one out of six (16.67%) BM calves had at least one diarrhea episode. Diarrhea occurred without general impairment of clinical conditions  . Diarrhep = 0.29). At Wk1, the difference in estimated weight was significant (p < 0.05) and remained so until the end of the trial (Wk8), when the BM and WM groups had estimated median weights of 85.24  and 69.99 , respectively.At Wk0, the calves enrolled in the BM and WM groups had estimated median weights of 45.41  and 41.94 , respectively. The difference in weight between the two calf groups at the beginning of the trial was not statistically significant , with an average of 107,564 reads per sample . After quality filtering, 1,438,378 sequences were removed, leaving 6,306,292 sequences for subsequent analyses . p < 0.05) for all alpha diversity indices, indicating a substantial negative impact on the fecal microbiota diversity that persisted for at least 2 weeks after removing the antibiotic-containing WM from the diet. Equitability and Simpson evenness were significantly different also at Wk1 and Wk2 (p < 0.05), respectively.p = 0.0069505, from PERMANOVA between time points, 999 permutations). Concerning beta diversity between treatments at each time point, the BM and WM groups were separated at Wk4  opinion paper, feeding calves with milk containing antibiotic residues presents a significant risk for the development of antimicrobial resistance . AnotherA relevant advantage of this study was the administration of standardized colostrum, TM, and WM, together with WM characterization in terms of antibiotic concentration and nutrient content. In this way, there were no differences in colostrum quality among calves or calf groups, and the composition and antibiotic content of WM remained the same throughout the trial. However, some limitations were also present.For ethical and practical reasons, the number of calves enrolled in the trial was limited to six per group, and calves were enrolled sequentially, first in the BM and then in the WM group. To offset these issues, the trial was carried out in a reduced time frame, and stringent statistics were applied to highlight the most relevant differences between the groups.We observed some differences in BM and WM calves' fecal microbiota at the beginning of the trial. Newborn calves have an unstable microbiota, as in the first day of postnatal life, the microbial community's relative composition changes dramatically . TherefoAnother point to consider is that WM from cows with mastitis likely had a different milk microbiota in itself than BM. Therefore, the different microbiota in calves fed with WM could have resulted from the microbes being ingested ; the study design model used here did not allow us to dissect the effect of drug residues from other factors that differed between WM and BM, such as milk composition and milk microbiota effect on fecal microbiota . FurtherThe 16S rRNA gene analysis approach provides information only on bacteria. However, the gut microbiota also includes archaea, protozoa, viruses, algae, and fungi that play crucial roles and participate in maintaining eubiosis , 59. ForOur study was carried out on male calves for animal value issues and ethical aspects due to female calves' more extended life expectancy. Long-term effects in the dairy farm are of interest mainly for what concerns female calves, and therefore gender effects may have to be evaluated more carefully. The breed might also play a role in resilience to intestinal microbiota perturbations , 65.Finally, first-generation cephalosporins are widely used for the intra-mammary treatment of clinical mastitis and are therefore one of the antibiotic classes most likely to be found in WM from cows with bacterial mastitis , 67. HowDuring the 2 weeks of WM feeding, we observed a significant increase in calf diarrhea incidence. Mitigation of pre-weaned calf mortality is a substantial challenge of the modern cattle industry, and enteric problems are among the major causes of newborn calf death , 70. WheWM feeding led to a dramatic loss in the fecal microbiota's alpha diversity compared to BM. The difference was already evident at Wk2 and highest at Wk4, both concerning richness and uniformity. Therefore, the adverse effects of WM in pre-weaned calves persisted and increased even under a diet with milk replacer containing probiotics integrated with pelleted starter feed, which should instead have led to an increase in the number of bacterial phylotypes in the calf gut . NotablyNumerous taxa showed significant changes in abundance in calves fed with WM vs. BM, starting from the beginning of the trial and up to 6 weeks after removing WM from the diet. The significant differences observed in the fecal microbiota of WM calves might result from the selective action of cefalexin on some bacterial groups, with a resulting alteration in the microbial equilibria resulting in dysbiosis. On the other hand, the significantly higher incidence of diarrhea in the first weeks of life, due to the elevated antibiotic concentration in WM, could have been responsible for disrupting the microbial ecosystem and the consequent incomplete recovery of the healthy stable state .Veillonella was already decreased in WM calves, in agreement with Van Vleck Pereira et al.  production and gut health . Written informed consent was obtained from the owners for the participation of their animals in this study.MP participated in the feeding trial, data analysis, data interpretation, and manuscript drafting. GS and AB contributed to the feeding trial, clinical monitoring of calves, sample collection, and clinical data analysis and interpretation. PC, BC, and FB contributed to the 16S data generation, analysis, and visualization. VB contributed to the bacteriological culture of milk, selection of cows, and data interpretation. PM and DP contributed to the study conception and design and data interpretation. MA contributed to the study conception, design and coordination, data interpretation and visualization, and manuscript drafting. All authors contributed to the revision and approval of the final manuscript.The 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.


Bifidobacterium animalis, Agave fructans, and symbiotic of both encapsulated on growth performance, feed efficiency, blood parameters, and immune status in broiler chickens, and to compare these with diets including antibiotic growth promoters and without additives.The present study was conducted to evaluate the effects of dietary supplementation with Bifidobacterium animalis (11.14±0.70 log CFU/g); SYM, a standard diet with B. animalis and Agave fructans.A comparative experimental study was carried out with 135 male Ross 308 broiler chickens. Each trial was divided into 5 equal groups. Control group (CON) received a standard diet without growth promoter; GPA, a standard diet with colistin sulfate and zinc bacitracin (0.25 g/kg of feed); PRE, a standard diet with 1% Agave fructans; PRO, a standard diet with A significant decrease in food consumption was found for the GPA, PRE, and SYM, compared to the CON group. The results show a better feed conversion index in PRE and GPA with respect to the CON group with the highest conversion index. Interestingly, the weight of the gastrointestinal tract shows a statistically significant difference between GPA and PRE groups. Moreover, the length of the gastrointestinal tract of the GPA group was less than the PRE group. In the total leukocyte count, there was a statistically significant increase in the GPA group compared to the CON, PRE, and PRO groups, and the heterophiles-lymphocytes index was lower in PRO. Regarding the cytokines, interleukin 10 (IL-10) decreased in PRO compared to CON and PRE, while IL-1β increased in the SYM group.Alternative treatments were shown to achieve similar productive results as growth-promoting antibiotics and showed improvement over diet without additives; however, they have immunomodulatory properties and improved the development of the gastrointestinal tract compared to the treatment of growth-promoting antibiotics. In broiler diets, antibiotics are used to improve growth performance and are known as growth-promoting antibiotics (GPAs). Also, they are used as a prophylactic therapy to prevent the development and transmission of diseases . It has Bifidobacterium animalis and Agave fructans are promoted more efficiently when both work together in the living system since the symbiotic relationship contributes significantly to health [Probiotics have been used to improve animal performance and maintain the normal microbiota of host animals. Their main action is a reinforcement of the intestinal mucosa barrier against adverse agents . Prebioto health . Symbioto health . The aimBifidobacterium animalis subsp. lactis (BLC-1) (11.14±0.70 log CFU/g) and 10 g of native Agave fructans  in a double emulsion. Development, production, and characterization of probiotic, prebiotic, and symbiotic capsules were performed and described by Juárez-Trujillo et al [The symbiotic was composed of The experimental protocol was performed following the guidelines of chapter 7.10 of the Terrestrial Animal Health Code of the World Organization for Animal Health  and the Bifidobacterium animalis (11.14±0.70 log CFU/g); treatment 5 (SYM), animals fed with a standard starting and ending diets with symbiotic capsules (11.14±0.70 log CFU/g+10 g Agave fructans). Food was offered ad libitum in poultry hopper feeders and the leftover was weighed each day to obtain the food consumed. Drinking water was also provided ad libitum daily in glass-bottom waterers.A comparative experimental study was performed with a total of 135 one-day-old male broiler chickens Ross 308 , with an average weight of 42.38±3.5 g. The birds were randomly distributed into five groups with 27 birds each. The broilers were housed in 15 experimental units consisting of a pen of 1 m×1 m each. All birds were administered with an initial standard diet  and finisher standard diet  with or without the following additives . TreatmeThe euthanasia of the broiler chickens was performed at the end of the experiment (49 days) using the cervical dislocation technique described in the “Manual for the welfare of broiler chickens” published by the National Service of Health, Safety and Agri-Food Quality of Mexico  and NOM-Growth performance was monitored during the initiation stage, finishing stage, and throughout the fattening. For total body weight gain, all broilers were weighed individually on day 0 and subsequently per week, subtracted the initial weight. The weekly food consumption was evaluated by adding the food consumed during all the days of the week. The feed conversion ratio (FCR) was calculated by dividing the grams of feed consumed for weight gain, during each stage and at the end of the experiment.On day 42, 10 broilers per treatment were randomly selected and euthanized, and the entire GIT, liver, gallbladder, and spleen were weighed. The GIT of all broilers was measured longitudinally.On day 42, two mL were collected from the wing vein for each bird into EDTA (5% ethylenediaminetetraacetic acid) tubes for the determination of hematological parameters. Hematocrit determination was performed by the micro-method . HemogloThe serum levels of the anti-inflammatory cytokines interleukin 10 (IL-10) and gamma interferon (IFN-γ), tumor necrosis factor-β (TNF-β), and pro-inflammatory cytokines TNF-β and IL-1β and IgA serum levels were quantified by ELISA kits following the instructions manufacturer at the 42 days. The kits were as follows: chicken IgA ELISA kit , chicken TNF-β , chicken IFN-γ , chicken IL-10 , chicken IL-1β .Data are presented as mean values with their standard errors. Data were checked for normal distribution with the Kolmogorov–Smirnov test. One-way analysis of variance was used to analyze the results and the differences between different groups were analyzed by Tukey’s multiple comparisons. It was considered at p<0.05 as significant (Statistica 8.0).During the initiation stage (1 to 21 days) there were no statistically significant changes for body weight gain the values are shown in In the same way as the weight gain, during the start stage (1 to 21 days) there were no statistically significant changes in food consumption. From the completion stage (beginning on day 21), a statistically significant decrease was found for GPA, compared to CON (p<0.05). Likewise, there was a statistically significant decrease for PRE and SYM to CON (p<0.05). All values are shown in The feed conversion did not show statistically significant changes in any of the stages or the total fattening. In the completion stage, the PRE treatment improved feed conversion by 14% compared to the CON.On the other hand, in the total leukocyte count, there was a statistically significant increase in GPA compared to CON and PRE and PRO (p<0.05). Moreover, there were statistically significant changes in lymphocytes and heterophiles in GPA with respect to CON and PRO, and SYM (p<0.05). The H:L ratio was very high in GPA compared to PRO and SYM. While, in the count of monocytes, eosinophils, and basophils there were no significant changes.For the biochemical parameters analyzed in blood, no statistically significant differences were found between treatments for glucose, cholesterol, or triglycerides (data not shown).Lactobacillus and limits the growth of Escherichia coli and C. perfringens [Lactobacillus species [Lactobacillus, Ruminococcus, and Clostridium genera were associated with improvement in productive parameters [This study aimed to evaluate the effect of a probiotic, prebiotic, and symbiotic encapsulated on productive performance, blood, and immune parameters in broilers, compared to colistin sulfate and zinc bacitracin and diet without additives. fringens . Convers species , which p species . In factrameters . Furtherrameters . These rrameters . Thus, trameters ,24.bifidobacteria and lactobacillus, has been shown to improve the health status and quality of meat in chickens infected with a pathogenic strain or subjected to stressors [Bacillus subtilis, Bacillus licheniformis, and Clostridium butyricum) and prebiotics , showing that the synergy increased daily weight gain and reduced the conversion rate. According to our results, other authors show that the growth performance in chickens does not increase with the dietary inclusion of probiotics and symbiotics compared to the control group [Moreover, the use of probiotics, mainly tressors ,26. Accotressors , the restressors , recordstressors  evaluateol group . Probablol group  a combinAgave fourcroydes with high concentrations of fructans did not cause adverse symptoms or significantly decrease hematological parameters [On the other hand, hematological parameters are used as indicators of health, and variations in these indicators can reflect bacterial, viral, parasitic, or fungal infections, as well as problems of intoxication, dehydration, clotting, or anemia . In generameters . Al-Saadrameters  reportedrameters . Regardirameters , where trameters  where thrameters  found thThe H:L index is recognized that it is a good indicator of acute and chronic stressors . Weimer Moreover, we did not find changes in the levels of biochemical parameters due to any of the treatments. According to this, Faramarzzadeh et al  showed tIn this study, we observed that the IL-10 and IFN-γ levels in the group administered PRO and GPA were lower. This may be due to the interaction that exists between the probiotic and the cells of GALT, generating inhibitory responses on the production of this interleukin. IL-10 plays an important role in viral and parasitic pathogenesis by suppressing the protective immune response of Th1 cells. IFN-γ expression has been associated with viral, bacterial, and parasitic infections; and it is well established that interferon plays a critical role in infection control and pathogen elimination . There aThe treatment of prebiotics, symbiotics, and GPA significantly reduced the daily food intake compared to the control group, demonstrating that prebiotics and symbiotics achieve similar productive effects as GPAs. However, prebiotics increases the weight and length of the GIT significantly compared to the group treated with GPA, demonstrating a better development with GIT with prebiotics than with GPA. Probiotic and symbiotic treatments decrease the heterophile-lymphocyte ratio and modify the levels of IL-10, IFN-γ, and IL-1β, suggesting that they may act as immunomodulators. These results suggest that Agave fructans is a novel additive that can be included in the diet of broilers to improve health parameters. However, more studies are suggested with different levels of treatment and also with different probiotic-prebiotic combinations to observe more benefits in broilers.