Permeation assays are important for the development of topical formulations applied on buccal mucosa. Swine buccal and esophageal epithelia are usually used as barriers for these assays, while frozen epithelia have been used to optimize the experimental setup. However, there is no consensus on these methods. In transdermal studies, barrier integrity has been evaluated by measuring electrical resistance (ER) across the skin, which has been demonstrated to be a simple, fast, safe, and cost-effective method. Therefore, the aims here were to investigate whether ER might also be an effective method to evaluate buccal and esophageal epithelium mucosa integrity for in vitro permeation studies, and to establish a cut-off ER value for each epithelium mucosa model. We further investigated whether buccal epithelium could be substituted by esophageal epithelium in transbuccal permeation studies, and whether their permeability and integrity were affected by freezing at −20 °C for 3 weeks. Fresh and frozen swine buccal and esophageal epithelia were mounted in Franz diffusion cells and were then submitted to ER measurement. Permeation assays were performed using lidocaine hydrochloride as a hydrophilic drug model. ER was shown to be a reliable method for evaluating esophageal and buccal epithelia. The esophageal epithelium presented higher permeability compared to the buccal epithelium. For both epithelia, freezing and storage led to decreased electrical resistivity and increased permeability. We conclude that ER may be safely used to confirm tissue integrity when it is equal to or above 3 kΩ for fresh esophageal mucosa, but not for buccal epithelium mucosa. However, the use of esophageal epithelium in in vitro transmucosal studies could overestimate the absorption of hydrophilic drugs. In addition, fresh samples are recommended for these experiments, especially when hydrophilic drugs are involved. Oral administration is one of the most convenient forms of drug delivery, due to the ease of ingestion and convenience for the patient. However, using this route, drugs might be altered due to first-pass metabolism and enzymatic degradation in the gastrointestinal tract. Topical administration through the buccal mucosa (inside the cheek) is a promising administration route. Transbuccal delivery overcomes the undesirable aspects of oral administration, increasing the compliance of patients since this pathway is non-invasive, offers simple application, and the medication may be easily removed in cases of adverse effects or allergies ,2,3.2) [However, transbuccal topical administration has some disadvantages, such as the limited area available for absorption (200 cm2) ,5 and re2) . This ma2) ,7,8,9,10In vitro permeation assays are essential for evaluation of the performance and quality of newly designed formulations, considering the physicochemical characteristics of combinations of components and excipients tested during the development of topical formulations . In addiFor transbuccal or transmucosal formulations, human buccal tissues are not usually used, because they are difficult to obtain and approval by ethics committees is required. Swine esophageal and buccal epithelium mucosas have been demonstrated to have similar histological, structural, and compositional features as the corresponding human tissues ,14. FurtSince mastication might damage the swine buccal epithelium, and only small areas of this mucosa are available, studies have proposed the use of esophageal mucosa as a buccal mucosa substitute, due to its similar histology and lipid composition, as well as simpler preparation ,14. In aVerification of tissue integrity prior to permeation assays was not performed in most of the early studies involving buccal and esophageal barrier models ,15,19,20Skin integrity is typically assessed prior to transdermal permeation assays. Among the different methods available, measuring electrical resistance (ER) across the skin has been used since the 1990s ,23, and 2 [2, but increased dramatically for ER lower than 2.5 kΩ/cm2. Therefore, we hypothesized that ER could be used as a reliable method to select intact epithelium mucosa barriers for permeation studies.In 1991, De Vries and colleagues described the use of this method to evaluate the permeability of dermatome buccal mucosa samples (0.24–0.80 mm thickness), obtaining ER values varying between 1 and 2 kΩ/cm2 . Later, 2 . It was The aims of the present study were to determine whether buccal and esophageal epithelium mucosa integrity can be predicted by ER for in vitro permeation studies, as well as to establish a cut-off ER value for each epithelium mucosa model, in order to predict barrier integrity. The possibility of substituting buccal epithelium by esophageal epithelium in transbuccal permeation studies was evaluated, together with assessment of the effect of freezing for 3 weeks at −20 °C on the permeability and integrity of the epithelia.2HPO4, and 0.24 g/L of KH2PO4; 1×, pH 7.4) produced with chemical reagents from Dinâmica Química . Swine maxillae and esophagi were obtained from the Angelelli Ltd., a slaughterhouse in Piracicaba , which was certified by the São Paulo State Department of Agriculture and Supply (SIF 2259). Permeation assays were performed in vertical transdermal Franz diffusion cells . Analyte quantification was performed using a high-performance liquid chromatography (HPLC) system coupled to an automatic collector and controlled with Surveyor ChromQuest 5.0 software .Lidocaine hydrochloride, acetonitrile, and ammonium hydroxide (HPLC grade) were purchased from Sigma-Aldrich . Carbopol, propylene glycol, methylparaben, triethanolamine, and glycerin were obtained from Galena Química e Farmacêutica . Ultra-purified deionized water  was used for the preparation of aqueous solutions, including phosphate-buffered saline (PBS)  esophagi and maxillae were transported to the laboratory in PBS solution, within 30 min after slaughter. The esophageal and buccal epithelia were prepared according to the methods described by Diaz Del Consuelo et al. [Swine  was isolated from the maxillae A,B and iEach experiment was performed with tissues from at least three different animals. The buccal and esophageal mucosas were separated from the adjacent tissues using a scalpel blade and immersion in a distilled water bath (60 °C for 2 min). The epithelium was gently detached from the connective tissue (lamina propria) with a round spatula, followed by initial visual inspection of the epithelium samples to detect macroscopic tissue irregularities. Half of the specimens were used immediately after preparation (fresh tissue), while the remainder were stored for three weeks at −20 °C.The fresh and frozen esophageal and buccal epithelium samples were examined to confirm the epithelium type and its integrity after the preparation and freezing processes.The epithelium samples were fixed in 10% buffered formalin, dehydrated using a series of increasing ethanol concentrations , and subsequently embedded in paraffin and cut to 5 μm thicknesses using a microtome. Tissue samples were stained with hematoxylin–eosin prior to analysis using an optical microscope  coupled to a digital color camera (Leica DFC280) .2 and receptor compartment volume of 7.0 mL containing PBS at 37 °C. The epithelium was positioned in the Franz cell so that the basal layer was facing the receptor compartment.After tissue preparation and initial visual inspection, ER assays were performed ,23,28. TThe donor and receptor compartments were both filled with degassed PBS buffer and were fitted with Ag/AgCl electrodes connected to a Keysight 33220A signal generator  and an ET-2053DMM digital multimeter , respectively. After allowing 1 h for electrolytic equilibrium under magnetic stirring (350 rpm) in a water bath at 37 °C, alternating current at 100 mV (rms) and frequency of 10 Hz was applied for resistivity evaluation. In order to avoid the effects of temperature, lipid composition, and barrier structural integrity , all theP is the potential difference of the system (mV), I is the measured current (µA), and A is the area (cm2).The electric current generated was measured by the multimeter in the receptor compartment, enabling the calculation of ER according to Ohm’s law (Equation (1)):2 were used in the subsequent stages of the experiment [Tissues exhibiting initial ER equal to or greater than 3 kΩ/cmperiment .n = 20) were submitted to ER measurements. Following this, the permeation areas of half of the specimens of each tissue were delimited prior to freezing .2 were wrapped in a cellulose filter , moistened with PBS, and then wrapped in aluminum foil. The samples were packed in a sealed plastic bag and stored for three weeks in a freezer at a controlled temperature of −20 ± 1 °C.For storage, half of the specimens initially presenting ER greater than 3 kΩ/cmLidocaine hydrochloride (LDH) was the drug model used for the permeation assays. A 5% lidocaine hydrogel was prepared as described previously .The experiments were performed with the fresh or frozen swine tissues, using a vertical Franz-type diffusion cell . ImmediassJ) was mathematically calculated according to Fick’s first law (Equation (2)). This mathematical model is well known for use in the analysis of permeation assays and for comparisons of studies [Qt is the difference of the amount of drug permeated, ∆t is the difference of the measurement time points (min), and A is the permeation area (cm2).During 1 h permeation assays, the temperature and stirring conditions were the same as in the electrical resistivity assays. At 10 min intervals, aliquots of solution 300 μL) were withdrawn from the receptor compartment for analysis of LDH by HPLC (described below). The same volume of PBS was added to the receptor compartment, with a calculation of the dilution. After quantification by HPLC, individual graphs  were obtained by plotting the amount of LDH accumulated in the receptor compartment versus time . The transport of LDH was analyzed according to a passive diffusion model, as commonly performed in studies of permeation across buccal and esophageal mucosas ,14,30,310 μL were studies .(2)Jss=v/v) mixture of acetonitrile and 25 mM NH4OH, adjusted to pH 7.0 with H3PO4 solution, at a flow rate of 1.2 mL/min. A reversed phase column  was used, for which the injection volume was 20 µL, and the detector wavelength was 220 nm. The specificity of the method was checked using a triplicate calibration curve on three different days, with different concentrations of lidocaine solution. The limits of detection and quantification were 0.24 and 0.80 mg/mL, respectively.Analysis of LDH was performed by HPLC, following a previously validated method with slight modifications . Brieflyt-test with Welch’s correction. The data for the cumulative amounts of LDH permeated after 1 h (1hQ) were submitted to ANOVA with Tukey’s test.The D’Agostino–Pearson and Bartlett tests were used to assess data normality and the homoscedasticity of variances, respectively. The data for the amounts of LDH permeated over time were submitted to linear regression analysis. The resistivity and steady-state flux results were evaluated using Brown–Forsythe ANOVA and an unpaired rP) test. All the statistical analyses were performed using GraphPad Prism 7.0 software , considering a significance level of 5%.Correlations between ER and the steady-state flux, considering each storage condition and tissue, were analyzed using Pearson’s correlation (As shown in p = 0.4402). Similarly, there was no difference between the values for these epithelia after freezing (p = 0.11) or after perforation of the fresh epithelium (p = 0.6788). However, lower ER values were observed after perforation of the frozen esophageal epithelium (p = 0.0121), compared to the buccal epithelium in the same condition  and the buccal epithelium   .p = 0.0008) and frozen (p < 0.0001) esophageal epithelium, compared to the buccal epithelium. Higher permeation of LDH across both esophageal and buccal epithelia was observed after freezing, compared to the corresponding fresh tissues   .ssJ) determinations were between 15 and 60 min in all cases. For both the fresh and frozen tissues, the permeation profile regression analysis showed that ssJ and the total amount of LDH permeated (1hQ) were higher for the esophageal epithelium, compared to the buccal epithelium .The calculated permeation parameters are summarized in ssJ and 1hQ for both the esophageal epithelium (p < 0.05 for both parameters) and the buccal epithelium (p < 0.0001 for both parameters), with approximately two-fold higher LDH fluxes across the frozen tissues  or the frozen buccal mucosa . However, the frozen esophageal mucosa  and the fresh buccal mucosa  showed strong inverse correlations between ER and the steady-state flux.This study investigated whether ER measurement could be used as an effective method to evaluate the integrity of esophageal and buccal epithelia employed in in vitro transmucosal permeation studies. It was decided to only use mucosal epithelium obtained by a heat-separation process, since this procedure has been demonstrated to have no impact on the morphology and permeability of oral cavity epithelium or esophagus mucosa ,15,21. H2) was based on previous findings, demonstrating that the permeation of hydrophilic drugs across esophageal epithelia remained the same for ER values between 2.8 and 12.7 kΩ/cm2 [To assess the reliability of this method, first a comparison was made between the ER values for pre-selected intact tissues and intentionally damaged mucosas. The pre-selection of intact epithelia  and buccal mucosa . It has been observed previously that the conductance values of damaged skin can also be quite variable, despite creating the same number of holes [Cubayachi and colleagues  obtainedof holes . In the Analysis was also made of correlation between the steady-state fluxes of LDH across the fresh or frozen epithelium samples and the corresponding ER values prior to the permeation experiment. LDH was chosen as a model hydrophilic drug for the permeation assays, because it has been used in ER measurements of ion transport across the skin, involving a hydrophilic transport pathway ,37,38. H2) was used as a barrier, the permeation of LDH was independent of ER, since no significant correlation was observed between these variables. However, when the same barrier was submitted to the freezing and storage process, there was a strong inverse correlation between ER and the steady-state flux of LDH, indicating that the permeation was influenced by ER. Similarly, previous work found that when ER was lower than 2.5 kΩ/cm2, greater amounts of lidocaine and prilocaine permeated across swine esophageal epithelium [The analysis of correlation between the steady-state flux and ER showed that when the fresh esophageal epithelium , ER ≥ 3 kΩ/cmAdditionally, freezing and storage may have a significant effect in relation to tissue damage. Therefore, it would be preferable to determine the permeability of hydrophilic compounds using fresh tissues.


Methods: Physiological, subjective, and cognitive performance data were collected from people working in ambient conditions characterized by the same thermal stress but different solar radiation levels. Results: People working under the sun were more likely to experience dizziness, weakness, and other symptoms of heat strain. These clinical impacts of sun exposure were not accompanied by changes in core body temperature but, instead, were linked with changes in skin temperature. Other physiological responses  were also increased during sun exposure, while attention and vigilance were reduced by 45% and 67%, respectively, compared to exposure to a similar thermal stress without sunlight. Light-colored clothes reduced workers’ skin temperature by 12–13% compared to darker-colored clothes. Conclusions: Working under the sun worsens the physiological heat strain experienced and compromises cognitive function, even when the level of heat stress is thought to be the same as being in the shade. Wearing light-colored clothes can limit the physiological heat strain experienced by the body.Background: A set of four case-control ( The long-term health effects of sun exposure have been extensively studied, particularly in relation to skin cancer and cataract, but there is little evidence-based knowledge on the acute impacts of sun exposure. For instance, a laboratory study showed marked negative effects of solar radiation on human cognitive performance , but we Agriculture and construction include the vast majority of employees exposed to the sun ,3 due toBased on these important knowledge gaps for addressing sun exposure and heat stress emergencies, the goals of this article are to provide health advisors and medical readers with evidence-based information on (i) the effects of sun exposure on worker physiology and cognition and (ii) a practical and economically feasible protection strategy for the most vulnerable individuals.The goals of this paper are achieved by presenting relevant findings from a series of studies carried out in different industries and countries that include two large field trials, one randomized controlled trial in laboratory settings, and two field interventions. A set of four interconnected studies was conducted as follows:Study 1: The effects of solar radiation on the psychophysical stress experienced by workers who perform manual labor in construction and agriculture. The aim of this study was to investigate the effects of solar radiation on human psychophysical stress during actual work shifts in the heat.Study 2: The effects of solar radiation on physiological responses and cognitive function at rest and during physical work. The aim of this study was to perform a controlled, laboratory-based evaluation of the effects of solar radiation on human physiological responses and cognitive performance at rest, during physical work, and post-work recovery by comparing indoor  and outdoor  environments characterized by the same thermal stress.Study 3: Identifying factors increasing the adverse effects of sun exposure experienced by agriculture and construction workers. The aim of this study was to investigate possible factors exacerbating the effects of sunlight-induced thermal strain by investigating workers’ behavioral habits during actual work shifts in occupational settings.Study 4: Interventions to mitigate the sunlight-induced heat strain experienced by people who work in agriculture and construction. The aim of this study was to test interventions to mitigate the sunlight-induced heat strain experienced by workers who work in agriculture and construction.Study 1: Detailed information on Study 1, including information about the physiological data we collected as well as supplemental tables and figures, is presented in Study 2: Detailed information on Study 2, including information about the physiological data we collected as well as supplemental tables and figures, is reported in Study 3: Detailed information on Study 3 is overviewed in Study 4: Detailed information on Study 4 is outlined in Sun exposure increases the physiological heat strain experienced by workers and compromises their cognitive function, even when the level of heat stress is thought to be the same as being in the shade. An overview of our findings is presented in 2 to 1043 W/m2). Of the 396 h, 108 (27.3%) were successive hours. Solar radiation levels were positively associated with mean skin temperature (Tsk)  as estimated by gastrointestinal temperature , mixed (161–320 W/m2), and outdoor (>320 W/m2) environments  = 57.791, p < 0.001).A group of 109 workers  participn levels . On the .035; sk  and labo.035; sk  were idesk above 36 °C , having a Heat Strain Score Index greater than 18 (indicating dangerously high risk of experiencing heat strain) was 3.61 times more likely when working outdoors as compared to indoors . Even mot strain ,15) was sk and Tcore were increased) related to Tsk and Tcore  was monitored for a total of 112 work shifts over a period of three months. We identified that more than two-thirds (68.8%) of the monitored workers wore dark-colored clothes during work under the sun. This was recognized as an important factor increasing the thermal strain experienced by workers. It is important to note that this is against prevailing recommendations and may reflect the lack of knowledge of workers and employers regarding occupational health and safety.2; and construction: ~200 ± 150 W/m2). Moreover, task analysis identified that workers performed similar manual work (agriculture (BAU: 194 ± 54 W/m2 vs. CLO: 189 ± 53 W/m2) and construction (BAU: 96 ± 17 W/m2 vs. CLO: 86 ± 17 W/m2)) during both scenarios. We identified no significant differences in the Tcore of workers between the tested scenarios (agriculture (BAU: 37.3 ± 0.3 °C vs. CLO: 37.2 ± 0.3 °C) and construction: (BAU: 37.3 ± 0.2 °C vs. CLO: 37.4 ± 0.2 °C)). Importantly, although workers were exposed to environments characterized by the same thermal stress and doing the same labor, workers donning white uniforms experienced a reduced level of physiological heat strain  was 13% and 12% lower during the white clothing scenario compared to the business-as-usual scenario in agriculture and construction, respectively.A group of six agriculture  and 41 construction  workers participated in the study . The twoTo investigate the physiological and health impacts of sun exposure on workers performing jobs/duties outdoors, a series of four separate but interconnected studies were conducted across different industrial sectors and countries. A large-scale occupational field trial was conducted in Qatar involving 109 construction and agricultural workers. From the collected data, we compared individuals working in the shade versus those working under the sun. We also isolated 396 work hours characterized by the same high level of heat stress  but very different levels of sun exposure: 33% were in the shade, and 67% were under the sun. We found that people working under the sun were four times more likely to experience dizziness , three times more likely to report weakness, and twice more likely to suffer other symptoms of heat strain , compared to performing the same work in the shade under the same level of heat stress. These clinical impacts of sun exposure were not accompanied by changes in core body temperature but, instead, were linked with changes in skin temperature, which was 10 times more likely to be at levels indicative of heat strain (>36 °C) when working under the sun.Skin temperature is an important parameter linked with both physiological and psychophysical stress ,15,17. ATo delve deeper into the physiological and cognitive impacts of sun exposure, as well as to better position the findings of our field experiments, we conducted in Greece a randomized controlled trial wherein seven healthy individuals were monitored during rest, moderate-intensity physical work, and post-work recovery inside a climate-controlled chamber. We compared values when participants were under the sun versus in the shade in temperate (20 °C WBGT) and hot (30 °C WBGT) ambient conditions. This study confirmed that sun exposure could elevate skin temperature without affecting core body temperature. Other physiological responses  were also increased during sun exposure but to a smaller degree. More importantly, sun exposure reduced cognitive performance in both temperate and hot conditions, leading to a 45% reduction in divided attention  and a 67% reduction in vigilance tasks  during 112 full work shifts performed outdoors in Cyprus during summer and autumn. Overall, the findings from the third study showed that 68.8% of the studied outdoor workers wore dark-colored clothes. This is against prevailing recommendations and reflects the lack of education and training on heat-related aspects of occupational health and safety for both workers and employers. Dark-colored clothes have high absorbance of radiative heat, and it is likely that workers would benefit from changing to light-colored work uniforms to minimize the adverse effects of sun exposure.In an occupational field intervention study performed in Cyprus, we tested whether white clothes can be used as a practical and economically feasible strategy to limit the impacts of sun exposure in agricultural workers. We monitored six workers during two full work shifts characterized by temperate conditions. In the “business as usual” scenario, workers wore their preferred clothes. As expected, based on the results of the above-described observational study, these clothes were mostly black or dark-colored t-shirts and pants. In the “white clothes” scenario, workers were provided with white hats and t-shirts  and were instructed to wear light-colored pants of their own. The findings from the fourth study demonstrated that the change to white/light-colored clothing minimized the increase in mean skin temperature during work by 13% (corresponding to a reduction of 0.4 °C), despite that the participants worked at the same level of effort in the same outdoor conditions.In a second occupational field intervention performed as part of our above-mentioned occupational field trial in Qatar , we testMedical staff are often asked whether occupational injuries are caused by exposure to the sun per se or by other parameters of heat stress such as high temperature or humidity. The field studies and laboratory-based clinical trials that we conducted in different parts of the world under both temperate and hot conditions show that working under the sun worsens the physiological heat strain experienced and compromises cognitive function, even when the level of heat stress is thought to be the same as being in the shade. To limit these detrimental impacts of sun exposure in cases where no other cost-effective shading solutions are available, medical staff, as well as health and safety professionals, should advise outdoor workers to wear white or light-colored clothes and hats/helmets. These pale colors increase the reflection of heat from the sun and can limit the heat strain experienced by the body. This multi-country series of field, clinical, and intervention studies raises another important issue; guidelines and policies should consider sun exposure as an important modifier of occupational and public health, not as one mere physical element to be included in the calculation of heat stress. Occupational and public health guidelines should be adapted based on exposure to solar radiation, fueled by a much-needed estimation of the associated burden of disease and injury.


In equine medicine, PRP is commonly used to treat joint diseases, tendinitis, ligamentous lesions, and persistent endometritis. Jenny endometrium shows a high sensitivity to endometritis. There are important differences between donkey and horse blood characteristics. Several protocols to obtain horse PRP have been reported, but no protocols have yet been reported for obtaining donkey PRP. Our study shows that single-spin tube centrifugation at 133× g for two different centrifugation times (10 and 15 min). The PRP samples were evaluated by flow cytometry, and TGF-β1 (Transforming Growth Factor-Beta1) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). The 10 min centrifugation protocol resulted in a slightly greater release of TGF-β1 (6044.79 ng/mL), a 2.06-fold increase in platelet concentration, and a 15-fold reduction in leukocyte concentration when compared to the initial values. The 15 min centrifugation time resulted in a 2.44-fold increase in baseline platelet concentration, a reduction in WBC count by a factor of 20, and slightly lower TGF levels (5206 ng/mL). We conclude that both protocols are adequate for the obtention of PRP, and both may have an acceptable therapeutic potential for use in this species, although this needs to be further validated.The aim of this study was to standardize a simple, manual platelet-rich plasma (PRP) protocol in Catalonian donkeys using single-spin tube centrifugation as a treatment for jenny endometritis. The objective was to obtain a blood product with a moderate concentration of platelets  and a low WBC (White Blood Cells) concentration. Blood was drawn from six Catalonian donkeys using acid citrate dextrose (ACD) as an anticoagulant, and then processed by single centrifugation at 133×  Jenny endometrium shows high susceptibility to endometritis. Semen deposition induces an exacerbated inflammatory response, which is more pronounced than in mares ,2. JennyAfter infection, neutrophils destroy pathogens by phagocytosis and by degranulation, producing DNA complexes that combine with nuclear and cytoplasmic proteins to form web-like extracellular structures called neutrophil extracellular traps (NETs) . HoweverThe failure of uterine drainage mechanisms in a mare can induce persistent metritis. NETs persistence has also been associated with the development of progressive fibrosis. Prostaglandins, inflammatory cytokines, and proteases found in NETs , cathepsin G (CAT), and myeloperoxidase (MPO)) act as pro-fibrotic factors in equine endometrial fibrosis by inducing collagen type I (COL1) accumulation ,8,9,10.Despite endometrial susceptibility, jenny endometritis seems to have its specific particularities, especially in the progression towards endometriosis. Inflammatory cells such as PMNs and eosinophils differ in timing and abundance, and there are also significant differences in the collagen distribution between jenny and mare .Platelet-rich plasma is a platelet concentrate product that has been proposed as an essential agent to improve the regenerative capacity of damaged tissues , especiaSome of the most interesting molecules in PRP are the growth factors located within the platelet alpha granules (α). These factors play an important role in the modulation of inflammation and tissue repair since they have chemotactic properties and stimulate mitosis, proliferation, cellular differentiation , neovascOne of the limitations of PRP is the lack of standardization of preparation. Consequently, the color, volume, and concentration of platelets, leukocytes, growth factors, and other cell types may vary from one preparation to another, which can lead to uneven and sometimes unsatisfactory clinical results ,33,34,35The aim of this study was to standardize a simple, manual, and inexpensive method of PRP preparation in donkeys while obtaining enough volume for the treatment of jenny endometritis-endometriosis.This study was approved by the Ethics and Experimentation Committee of the Universitat Autónoma de Barcelona (CEUA/UFMG protocol number 294/2013). The experimental design is explained graphically in Whole blood was collected from six Catalonian jennies, with ages varying from 6 to 14 years. The donkeys showed a good body condition and were clinically healthy based on physical examination and a complete blood cell analysis. Jennies were grouped in a big paddock at the Equine Reproduction Service, Autonomous University of Barcelona . Animals were fed grain forage, straw, and hay, and water was provided ad libitum.The blood was collected through an aseptic puncture of the jugular vein, using a Vacutainer  system and a 23 G needle. A sample of the blood was taken and transferred to a 1 mL EDTA (Ethylenediaminetetraacetic acid) tube for a complete blood cell count (CBC), while the remaining blood was transferred to two 8.5 mL tubes (tubes 1 and 2), containing 1.5 mL of ACD (Anticoagulant Citrate Dextrose solution) anticoagulant  . In sequence, the blood was homogenized manually through slow inversions of the tube.g for two different centrifugation times, tube 1 being centrifuged for 10 min and tube 2 centrifuged for 15 min. In each tube, 2.0 mL of the plasma fraction was collected in a laminar flow chamber using an 18 G spinal needle . The tip of the needle was introduced until approximately 4 mm above the white blood cell layer (buffy coat) in order to recover the maximal number of platelets and the minimum possible amount of leukocytes.Several preparation protocols of PRP used in horses were tested in donkeys (non-published data), and the most interesting results were obtained via the simple tube centrifugation method. The following protocol was selected based on reproducibility and ease of preparation. The tubes were centrifuged (CTR) at 133× g to promote platelet sedimentation. After centrifugation, the supernatant was removed and immediately frozen and stored at −20 °C. These samples were submitted for ELISA testing to determine the TGF-β1 concentrations .Once the PRP was prepared, a 1.0 mL sample was submitted for a platelet count and evaluation by flow cytometry at the Veterinary Hematology Service of the Universitat Autònoma de Barcelona. The remaining PRP was then centrifuged at 1000× ® Siemens Medical Solutions; Fernwald, Germany), which is a flow cytometer with two laser beams, making it possible to contrast the same value and generate accurate and reliable results. The leukocyte, erythrocyte, and platelet panels were then evaluated. The platelet parameters studied included platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), mean platelet component concentration (MPC), platelet volume of distribution width (PDW), mean platelet dry mass (MPM), platelet dry mass distribution width (PMDW), platelet component distribution width (PCDW), concentration of large platelets (LAPLT), and platelet clumps (CLUMPS).Complete blood cell counts (CBCs) were performed by a hematological cytometer (ADVIA 120TGF-β1 (Transforming Growth Factor-Beta1) was measured by ELISA, using commercially available antibodies . The TGF-β1 concentration was determined using a mouse antigen-catching antibody specific for human TGF-β1 and a biotinylated chicken detection antibody, also specific for human TGF-β1. Human TGF-β1 recombinant patterns were also used to elaborate the standard curve. The use of this kit in horses has already been tested and approved in previous studies of this research group , but itsThe samples were activated to quantify the growth factors by ELISA. This activation triggered the release of latency-associated peptides and protein disulfide isomerase (PDI) and, consequently, the formation of the inactive TGF-β1 complex, secreted by the cells. The activation was performed in vitro by the addition of 100 µL of activation buffer  and urea 10 M  to the same volume of plasma, followed by homogenization and incubation at room temperature for 10 min. The pH was neutralized with 100 µL of neutralizing buffer /HEPES 1 M . We obtained different dilutions of the samples, activated with the dilution buffer , Tween 20 0.05% , in PBS .p-values were <0.05.The data were analyzed with commercial software  and expressed as means ± standard errors. Single-factor analysis of variance (ANOVA) was used for statistical comparisons, while the post hoc comparisons were analyzed through a Tukey’s test to determine the differences between means with normal distribution, as well as variables that presented a normal distribution after log (x + 1) transformation. The variables that did not present a normal distribution after the logarithmic transformation were analyzed through the Student’s t-test, and the post hoc comparisons were made through the Wilcoxon test. Differences were considered statistically significant when the p < 0.05) of PLT, MPV, MPC, PCDW, CLUMPS, WBC White Blood Cells), RBC (Red Blood Cells), and PMN. However, there were no differences in these values (p > 0.05) between the PRPs prepared with different protocols. The TGF-β1 concentration was greater in the protocol with a shorter centrifugation time  with success. The following estrus, both were inseminated and became pregnant.Two Catalonian jennies, 15 and 17 years old, with endometrial biopsies categorized as IIB in Kenney and Doig classification and presenting infertility for the last two years, were treated once. An intrauterine infusion was performed during estrus with 20 mL of PRP  increase, and decreases in MPC (density) and PCDW (platelet component distribution width). The 15 min protocol showed a slight decrease in TGF-β1 concentrations, and this is consistent with studies in other species which show that there may be variability in TGF beta yield even within the same individual at different sampling times [No studies were found in the literature regarding PRP preparation protocols in donkeys. This is the first study evaluating PRP preparation in donkeys, including the measurement of TGF-β1 concentrations with different protocols. The hematological parameters of whole blood were consistent with previous parameters described for Catalonian donkeys . The ADVng times .p < 0.05) in the platelet counts between centrifugation for 10 and 15 min. The tubes centrifuged at 133× g for 10 min presented a 2.06-fold increase in platelet concentration and a reduction in leukocyte concentration by a factor of 15 compared to initial values, whereas the tubes centrifuged at the same gravitational force for 15 min presented a 2.44-fold increase in platelet concentration and a 20 times reduction in leukocyte concentration. In accordance with the classification proposed by Dohan Ehrenfest et al. [There were no differences . The large individual variation, characterized by the standard error of the mean and coefficient of variation, may indicate the necessity of including a greater number of animals in future research.In the 10 min protocol, platelet activation was less apparent than in samples centrifuged for 15 min, as expressed by a lower number of CLUMPS, lower MPV, lower PCDW, and a higher MPC. However, the differences were not statistically significant , even with a lower platelet concentration, may be due to the higher concentration of viable non-activated platelets in this protocol compared to the 15 min protocol. It is necessary to conduct more studies regarding platelet activation since, according to some authors, a higher number of viable non-activated platelets is desired [The higher TGF-β1 concentration in the 10 min protocol ( desired ,45,46 inSeveral in vivo and in vitro experiments have shown beneficial effects of platelet-related products in the modulation of the inflammatory process and tissue healing. To optimize PRP composition and facilitate its preparation in other species, several methods have been described. Although the literature in PRP is extensive, the lack of evidence and consensus about what method or PRP composition to use in different diseases or tissue trauma remains. In general, manual methods intended for PRP production present lower costs, but may be time-consuming and require a strict aseptic technique. Since the method described here is based on one single spin with an acceptable end product that was validated clinically in a few animals, we have provided the first step to finding the ideal product in donkeys with a method that is simple and cost-effective.g for 10 min was appropriate to obtain supraphysiological concentrations of platelets with a significant and concurrent reduction in leukocyte content. With this protocol, the PRP also presented an acceptable concentration of TGF-β1 as a growth factor sentinel, possibly providing enough volume and therapeutic potential for use in damaged jenny endometrium. It should be noted that there are important differences between jenny and mare endometritis and endometriosis [The methodology employed for the PRP preparation in donkeys with single tube centrifugation at 133× etriosis , and thaetriosis ,47,48,49


Unit loads consisting of a pallet, packages, and a product securement system are the dominant way of shipping products across the United States. The most common packaging types used in unit loads are corrugated boxes. Due to the great stresses created during unit load stacking, accurately predicting the compression strength of corrugated boxes is critical to preventing unit load failure. Although many variables affect the compression strength of corrugated boxes, recently, it was found that changing the pallet’s top deck stiffness can significantly affect compression strength. However, there is still a lack of understanding of how these different factors influence this phenomenon. This study investigated the effect of pallet’s top-deck stiffness on corrugated box compression strength as a function of initial top deck thickness, pallet wood species, box size, and board grade. The amount of increase in top deck thickness needed to lower the board grade of corrugated boxes by one level from the initial unit load scenario was determined using PDS™. The benefits of increasing top deck thickness diminish as the initial top deck thickness increases due to less severe pallet deflection from the start. The benefits were more pronounced as higher board grade boxes were initially used, and as smaller-sized boxes were used due to the heavier weights of these unit loads. Therefore, supposing that a company uses lower stiffness pallets or heavy corrugated boxes for their unit loads, this study suggests that they will find more opportunities to optimize their unit loads by increasing their pallet’s top deck thickness. Historically, the distribution packaging industry has adapted the method of unitizing single, multiple, or bulk products on a solid platform to make the handling, storing, and transporting of these products easier . This arWhen designing a unit load, accurately predicting corrugated box compression strength is crucial to avoid package failure from the vertical compression forces during distribution and storage. Therefore, numerous studies have investigated the factors that affect the compression strength of corrugated boxes, including material properties ,7,8,9,10Wood pallet characteristics, such as pallet gaps and pallet overhang, have been included among the main palletization factors that affect box compression strength. In relatively recent years, researchers have endeavored to correlate pallet top-deck stiffness to corrugated box compression strength. Baker  and PhanMeanwhile, Baker  found thIn 2020, Quesenberry et al.  further Additionally, the corrugated boxes were made of a single board grade, two flute sizes, and two box sizes. In practice, many wood species with varying moisture content are available for pallet manufacturing; meanwhile, corrugated boxes are produced in multiple board grades and sizes. Nevertheless, there is an absence of studies investigating how these variations may change the effect of top deck stiffness on corrugated box compression strength.Therefore, the objective of this current paper is to investigate the effect of pallet top-deck stiffness on the compression strength of asymmetrically supported corrugated boxes as a function of currently under-studied variables, including initial top deck thickness, pallet wood species, box size, and board grade.This study consisted of two main sections: validation of the analytical pallet design software and unit load scenario analysis.The commercially available pallet design software Pallet Design System™ (PDS™) v. 6.2, created by NWPCA  was utilized to replace numerous physical experiments in this study. The box performance data predicted by PDS™ and that Quesenberry et al.  found weThe same designs of corrugated boxes used by Quesenberry et al.  were useQuesenberry et al.  simulateDuring software validation, the box load factors computed by PDS™ and the box compression strength factor derived from the thesis of Quesenberry  were comStep 1: Built a unit load in PDS™ using boxes and pallets previously described in Step 2: Set the top deck board thickness to the lowest level (9.5 mm).Step 3: Set the weight in the box to the load that will just fail the boxes (box safety factor of one) when the support condition is a single floor stack.Step 4: Report current box load factor when support condition is single floor stack.Step 5: Increased the top deck board thickness to the following levels .Step 6: Repeat steps 3 and 4 for each level of top deck board thickness.Step 7: Repeat the process for two flute sizes (B and C flute) and two box sizes.Box load factors were computed using PDS™ following the steps described below.CSF = Box compression strength factor.avgSCS = Average box compression strength when box corners are symmetrically supported on a rigid platform.avgACS = Average box compression strength when box corners are asymmetrically supported on the actual pallet.The unit load scenarios used to calculate the box compression strength factors were varied by two flute sizes, two box sizes, and four thickness levels.The box compression strength factor from Quesenberry’s study was calculated using Equation (1):t-test was conducted to see whether the difference between box load factors from PDS™ and box compression strength factors from the experiment were statistically significant or not. To confirm the normality assumption of the independent t-test, we also ran the Shapiro–Wilk test for each group separately. The similarities between the box performance data from PDS™ and the experiment were also assessed using the Pearson correlation coefficient. The Pearson correlation coefficient is a way to investigate linear dependence between two variables. The measured correlation coefficient (r) ranges between −1 and +1. When the r-value is −1, it indicates a strong negative correlation, while +1 indicates a strong positive correlation, and 0 means no relation. Both statistical analyses were conducted at a significance level of 0.05. The analyses were done using SAS JMP Pro 15® software .The independent The concept of a unit load cost optimization method that allows for corrugated boxes with decreased board grades by increasing the pallet’s top deck thickness was adopted from Quesenberry et al.  to modifThree sizes of RSC-style corrugated boxes were investigated to explore the effect of different box sizes. Three box sizes were chosen that would cover the entire top surface of the 1219.2 mm × 1016 mm pallet and create asymmetrically supported corners. The external dimensions were 203.2 mm × 304.8 mm × 254 mm , 406.4 mm × 254 mm × 254 mm (medium box), and 609.6 mm × 337.8 mm × 254 mm (large box). The boxes were organized in 4 × 5, 3 × 4, and 2 × 3 arrays for small, medium, and large boxes, respectively. Four layers of boxes were used for each unit load. Unit load configurations using the different box sizes are depicted in For the unit load scenario analysis, the most common size of GMA™ style wood pallet was used. The 1219.2 mm × 1016 mm GMA™ style stringer class wooden pallet is the most commonly used pallet design in North America ,29. The Initial top deck thicknesses were varied by four levels to explore which changes in deck board thicknesses would be required to reduce by one level the initial board grade specified for the corrugated boxes. The investigated initial top deck thickness levels were 9.5 mm, 12.7 mm, 15.9 mm, and 19.1 mm. However, unit load scenarios built with kiln-dried southern yellow pine (KD SYP) pallets were designed with 11.1 mm top deck boards, and this thickness was increased to 17.5 mm for the optimized design. This limitation was due to the availability of raw material sizes; only the 11.1 mm and 17.5 mm dimensions could be manufactured effectively.Wood species used for pallet construction were also varied. The wood species commonly used for pallet construction in the southeastern United States were selected. Selected wood materials were: green, high-density hardwood (Grn HD HW); green, low-density hardwood (Grn LD HW); green, southern yellow pine (Grn SYP); and kiln-dried, southern yellow pine (KD SYP). Green lumber contained 25% or greater moisture content, and kiln-dried lumber had a maximum of 19% moisture content.Several factors of the unit load were varied to identify the characteristics that could change the effect of the pallet’s top deck board stiffness on box compression strength. The factors evaluated were initial top deck board thickness, pallet wood species, box size, and corrugated board grade. The variable factors that relate to pallets are listed in The unit load cost optimization method adopted from Quesenberry et al.  was inveStep 1: Construct the unit load in PDS™.Step 2: Set pallet material as one of the listed wood species .Step 3: Set the top deck board as the lowest initial top deck board thickness (9.5 mm). In the case of KD SYP, always set the initial top deck thickness as 11.1 mm.Step 4: Set corrugated boxes as the higher ECT values in the selected range of board grade .Step 5: Determine the weight of the box that works to create a box safety factor of three for the double floor stacking condition.Step 6: Create a new unit load with the corrugated boxes made of lower ECT value from the selected range of corrugated board grade and apply the weight determined in step 5 .Step 7: Continuously increase the top deck thickness by 1.6 mm until the unit load again reaches the safety factor of three for safe operation. In the case of KD SYP, always increase the top deck thickness to 17.5 mm.Step 8: Report the total increase in the top deck board thickness required to achieve the required safety factor of three.Step 9: Repeat step 1 to step 8 after changing the pallet wood species.Step 10: Repeat from step 1 to step 9 after increasing the initial top deck stiffness level.Step 11: Repeat from step 1 to step 10 after changing the range of board grade .Step 12: Repeat from step 1 to step 11 after changing the size of corrugated boxes .Required steps in the analysis were as follows:The amount that the top deck thickness increased was categorized as one of three grades to make it easier to identify which scenarios had smaller or larger increases in top deck thickness: less than 12.7 mm (grade 1), 12.7 mm to 25.4 mm (grade 2), and beyond 25.4 mm increase (grade 3). For better visualization, a color-coding system was also applied; green for grade 1, yellow for grade 2, and red for grade 3. Grade 1 scenarios were considered as cases with high potential to apply the unit load optimization process. Grade 2 scenarios were considered cases that may be possible to apply the optimization method depending on the manufacturer’s circumstances. Because pallets made of deck boards thicker than 25.4 mm are unprecedented; grade 3 scenarios were considered unrealistic unit load designs.t-test showed that the difference between PDS™ and the experiment was not statistically significant (t(25) = −0.85, p-value = 0.40). The Shapiro-Wilk test confirmed that the normality assumptions were met . Furthermore, the Pearson correlation coefficient revealed a strong positive correlation, r = 0.911 , between box load factors from PDS™ and box compression strength factors from experiment results. In other words, the PDS™ and Quesenberry’s [Measurement of the box load factors and box compression strength factors on varied top deck thicknesses, box sizes, and flute sizes are presented in To investigate how different factors such as the initial top deck board thickness, pallet wood species, box size, and board grade effect the feasibility of optimizing the strength of the corrugated boxes by changing the stiffness of the pallets, researchers looked at the changes in the proportions of different grade scenarios in response to each variable factor.Furthermore, the proportion of the grade 1 scenarios for Grn HD HW was slightly lower (35%) than the other green lumber scenarios (40–42%). Since Grn HD HW does not have a limit on the level of top deck thickness increase, this could provide further evidence that the stiffness of the material affects the feasibility of the design scenario. Overall, the results indicate that the feasibility of using increased deck board thickness to lower the corrugated boxboard grade decreases when species with higher material stiffness are initially used to construct the pallets.Similar trends in the proportional changes of different unit load scenario grades were observed from the initial top deck thickness effect and the pallet wood species effect. Both results indicated a significant reduction in the potential to decrease board grade by increasing top deck stiffness when the pallet was initially designed with stiffer pallet wood material. In other words, this unit load optimization method is more effective when the unit load is initially designed using lower stiffness pallets.Overall, it was found that all investigated variable factors had an observable influence on the feasibility of using an increase in pallet top-deck stiffness to lower the board grade of the corrugated boxes. Unit load scenarios to which it was more feasible to apply the unit load cost optimization method were observed as the initial unit load was designed with less stiff pallet top-deck boards; either thinner top deck boards or lower density wood species. For box-related variables, unit loads of smaller-sized boxes, unit loads with a smaller range of board grade reduction, and unit loads with higher initial board grades all created more favorable situations on which to apply the unit load optimization method due to the heavier weight of these unit loads.Only a standard GMA™ style, stringer class, wooden pallet design was investigated in the study.PDS can only run analysis up to 38.1 mm top deck board thickness, so the scenarios requiring top deck boards thicker than 38.1 mm were not simulated. Therefore, the color grading system was applied to show the comparison between these different scenarios.Due to the functional limitations of PDS™ regarding top deck thickness increases, the correlation between pallet stiffness (kg/mm top deck deflection) and the amount of wood material that needed to be added was not investigated.The benefits of increasing a pallet’s top deck thickness to reduce the corrugated board grade diminish as the initial stiffness of the pallet increases.There were more opportunities to optimize unit load designs when the ECT values between the different board grade levels were lower.There were more possibilities of decreasing board grade when the initial board grade was higher, and/or when the box size was smaller, mostly due to the heavier weight of these unit loads, which caused greater pallet bending. Pallets made of Kiln-Dried Southern Yellow Pine are less likely to be able to be optimized using the investigated methods because of the limited deck board thicknesses that can be cost-effectively manufactured from the available raw materials.The key findings of this study were as follows:Therefore, this study suggests that companies that use low stiffness pallets or have unit loads of heavy boxes could have more opportunities to optimize their unit loads by increasing the top deck thickness of their pallets.The study also revealed that changing the top deck board stiffness cannot be done without considering the effects of other factors such as initial top deck board thickness, pallet wood species, box size, and board grade. Therefore, the unit load optimization process that reduces corrugated board grade by increasing top deck stiffness needs to be a holistic process.The next phase of the project will focus on investigating whether the increase in pallet top-deck stiffness and the resulting reduction in corrugated boxboard grade can create an environmentally beneficial scenario.


These findings can benefit the development and the optimisation of industrial oat baking processes.Whole grain oat has become an increasingly popular baking ingredient. Still, oat baking poses many industrial challenges because the baking quality criteria have not been set for whole grain oat flours, and cultivar variation remains unknown. We aimed to assess the baking quality variation of twenty whole grain oat cultivar samples, and to identify the factors that caused the variation. It was hypothesised that by optimising the water absorption of the dough  by test baking method, the best baking potential could be achieved for all oat cultivar samples. The baking trials were conducted as whole oat baking, without wheat or gluten additions. In most of the samples, good baking quality was obtained by dough yield optimisation. The highest specific volumes (1.9–1.93 mL/g) and best crumb properties were achieved in the samples with the highest optimal dough yields, 205. However, baking quality varied, as all samples could not be baked with good quality at high dough yields. Additionally, small median particle size and high fat content of the oat flours were related to good baking properties of whole grain oat at optimised dough yield ( Whole grain oat has gained great popularity as a healthy baking ingredient. Whole grain oat is rich in soluble fibre, β-glucan, and for example, the average β-glucan content was about 4% in oat cultivars grown in Finland . Oat β-gLack of gluten and high amount of β-glucan make whole grain oat special among the bread-making cereals, and oat baking differs largely from wheat baking. Wheat gluten is capable to produce a dough that retains gas and produces an aerated bread structure, while non-wheat breads tend to remain dense and low in volume . In wheaWhole grain oat baking has got little attention in the literature, since most oat baking studies have been conducted as mixed oat-wheat baking ,12. It hThe objective of this study was to assess the variation in the baking quality of twenty whole grain oat cultivar samples that were kilned and milled in similar conditions. The oat samples were grown in Finland in 2018–2019. It was hypothesised that the baking quality of oat differs among oat cultivar samples, as their physicochemical properties differed. We aimed to assess the range of the variation in baking quality, and to identify the factors that caused the variation. Baking qualities were evaluated after dough yield optimisation by test baking method, so that the optimal baking quality could be achieved in all oat cultivar samples.Whole grain oat flours representing 20 oat cultivar samples were studied, and the samples were obtained from Boreal Plant Breeding Ltd., Jokioinen, Finland; Peltosiemen Ltd., Forssa, Finland; Vääksyn Mylly Ltd., Vääksy, Finland; Plantanova Ltd., Ruukki, Finland, Raisio plc, Raisio, Finland, and Lantmännen Agro Ltd., Vantaa, Finland. This study was a part of a larger study, the OatHow project. The basic chemical properties of the oat cultivar samples were published previously in the project , and con50), the means of the particle diameters , and the particle diameters, that 90% of the particles are smaller (D90) were measured. Moreover, the moisture contents  and cenDough yields  were optimised by test baking method separately for each oat cultivar sample. In test baking trials, the straight dough baking process was developed and monitored. The dough size of 400 g for individual loaf bread was selected, as the bread size was suitable to evaluate the crumb properties, such as porosity and bread staling during storage, and the doughs did not exceed the baking pans during proofing and baking. The same proofing and baking times were suitable for all the samples, as the bread structures did not collapse during proofing, and the bread crumbs of all cultivar samples were mature after baking, without burning of the crust. It was observed that dough yield optimisation had a major influence on the baking quality. Thus, similar proofing and baking conditions were used for all the samples. Dough yields were considered optimised for the oat cultivar sample when the dough was workable by hand, the overall appearance of the bread was good, the crumb had even porosity, and there was no detectable rawness in the bread crumb.The straight dough baking recipe contained whole grain oat flours (at 14% moisture content), an optimised amount of tap water , syrup (4% of fb), baker’s yeast (3% of fb), salt (2% of fb), and psyllium (2% of fb), and the total weight of the dough batch was 1.5 kg in test baking trials. Psyllium was mixed with half of the dough water and left to set for 10 min. Then, all ingredients were mixed at low speed  and then at high speed  with a mixer with a paddle attachment . The dough rested for 15 min at room temperature before baking. Dough pieces of 400 g were baked by hand, placed in oiled baking pans (18 cm × 6 cm × 8 cm), and proofed at 35 °C (100% RH) for 30 min . The breads were baked in a convection oven at 205 °C for 30 min, with 20 s of steaming at the beginning . The breads were allowed to cool down for 60 min before evaluation.To evaluate the baking quality and storage stability of each sample, the oat cultivar samples were baked at optimised dough yield in three replicate doughs. Apart from an increase in dough size (from 1.5 kg to 5 kg), the recipe and the baking process followed the same practice as in test baking trials. The proofing and baking conditions were monitored at increased dough size, and it was observed that the process remained suitable. After 60 min from baking, the breads were weighed to measure the bake loss. Specific volumes of the breads were measured with a laser scan, VolScan Profiler .Bread qualities were evaluated by two oat baking expert assessors, who conducted the baking trials, and they were the two first authors of this study. In the test baking method, the baking quality evaluation includes instrumentally measured bread quality  but additionally, it includes the test baker’s expert assessment of the quality parameters and quality defects that cannot be measured instrumentally, such as unevenness and rawness of the bread crumb , taste faults (bitterness), and mouthfeel faults (hardness or stickiness). In test baking, the quality can be assessed properly only when both these aspects are considered.Bread quality evaluation by the expert assessors was based on fulfilling the set quality criteria and detection of the quality defects in the breads. Bread crumb structure (evenness of the porosity), bread shape, taste, and mouthfeel were rated according to set quality ratings . Bread crumb structure was rated excellent (3) if the crumb had an aerated structure and even porosity A. SingleBread crumb texture was analysed with a two-bite compression test  after 1, 2, and 3 days of storage at room temperature. A 5 kg load cell and a cylindrical probe with a diameter of 36 mm were used in the analysis. Four 25 mm thick slices were cut from each replicate bread, and the crust was removed carefully with a knife. The bread crumb sample was placed under the probe and compressed twice into 40% deformation at 5 mm/s speed, with a 5 s rest between the compressions. Bread crumb hardness was measured as the peak force (N) during the first compression. Staling rate was measured as the increase of crumb hardness (N) between storage days 1 and 3.Dough consistency was measured from the oat doughs at optimized dough yield with backward extrusion method, with a 5 kg load cell . Five replicate dough samples were compressed into 50% deformation with a disc probe (diameter of 35 mm) at 1.5 mm/s speed in a cylindrical container (diameter of 50 mm). The container was filled with the dough sample to a height of 40 mm, and the sample surface was oiled to prevent sample adherence to the probe. The dough samples were prepared for the analyses similarly to the test baking trials, but without yeast. Dough consistency (N∙s) was measured as the area between the force and the time axes during compression.p < 0.05.In the tables and figures, the average values of the measurements are presented. All analyses were performed at least in triplicate. The error bars and error values represent standard errors of the means (SEM). Correlation analysis was conducted with Pearson’s correlation coefficient . Analysis of variance (ANOVA) was done with Tukey’s HSD test . Statistical analyses were conducted at a significance level of 50) varied between 112–204 μm, average particle diameters  between 229–316 μm, and D90 values between 606–826 μm. WHC of the oat flour samples varied between 1.08–1.45 mL/g.Particle sizes and centrifugal WHC of the flours varied greatly between the oat cultivar samples . Median Centrifugal WHC of the oat flour samples were measured to predict the optimal dough yield in test baking trials, but it was observed that these properties were not related to each other. This was also confirmed in correlation analysis . Thus, tThe importance of the dough yield optimisation was clearly demonstrated as in most of the samples, good baking quality was obtained by dough yield optimisation. However, oat bread samples varied greatly regarding their overall quality, crumb structure, and bread shape A–D. Highp < 0.05) variation between the samples , as expected, but also with high bake loss (p < 0.001), high specific volume (p < 0.001), and soft crumb structure (p < 0.01). High dough yield, low dough consistency, and high bake loss were favourable in oat baking, since they correlated with high specific volume (p < 0.001) and soft crumb structure (p < 0.01). Bread crumb hardness correlated strongly between the storage days one and three (p < 0.001), indicating that the initial crumb hardness mainly determined the crumb hardness during storage. Low dough consistency, high bake loss, and high specific volume correlated with the low staling rate of the bread (p < 0.05).High dough yield correlated with low dough consistency  correlated with more than one baking quality parameter. High fat content of oat flours correlated with low dough consistency (p < 0.05), high specific volume (p < 0.01), and high bake loss (p < 0.01). Small median particle size (D50) correlated with high dough yield and high bake loss (p < 0.05), and low crumb hardness (p < 0.01). All particle size parameters  correlated positively with the staling rate (p < 0.05), indicating that the larger particle size was related to the greater staling rate of the bread. WHC and protein, starch, ash, and total dietary fibre contents of the oat flours did not correlate with any of the baking quality parameters (p > 0.05). β-glucan content of the oat flours showed only a weak correlation with crumb hardness, and only after one day of storage (p < 0.05).Among the physicochemical quality parameters of the oat flour samples, only the fat content and the particle size of oat flours  was a sample-specific trait, and too high and too low dough yields decreased the baking quality. However, high dough yield or low dough consistency could not be used alone for optimisation, as all samples could not be baked at high dough yields with good quality. In addition, optimal dough yield could not be predicted from the physicochemical quality factors of the oat cultivar samples, as only the median particle size correlated with optimised dough yield. Test baking is considered a time-consuming optimising method, but there is no other method to optimise oat baking quality, where both dough workability and bread quality are included. In wheat baking, it has been reported that the differences in bread volume became apparent during oven baking and could not be detected in earlier stages of baking ,20. In oIn our test baking trials, too high dough yields were eliminated as the doughs were too sticky to handle, or they caused impaired bread crumb structure. According to Eliasson & Larsson , decreasp < 0.001). The role of water evaporation during baking in bread structure formation has been discussed also previously [High optimal dough yield was beneficial in whole grain oat baking, and the advantages of high water content in the dough have been demonstrated also earlier in gluten-free baking ,10,21. Peviously ,23,25.p < 0.001), and thus, high dough yield was related to softer bread crumbs. Also earlier, high water content in the dough has been reported to decrease bread hardness in gluten-free baking [p < 0.001). Thus, the greatest specific volumes led to the softer crumbs , whereas the hardest breads had the lowest porosities and the tightest structures in the crumb. Hence, gas cell stabilization was also related to the oat bread structure and texture.In our study, high dough yield correlated negatively with bread crumb hardness , bread specific volume , and bake loss . Previously, the positive baking properties of oat oil have been demonstrated in wheat baking [Among the chemical data of the oat cultivar samples, only the fat content of the oat flour samples correlated with the baking quality parameters; with dough consistency . Previously, the baking quality of gluten-free rice bread increased after β-glucan addition at low concentrations (1–1.3% of the flour weight) while at higher concentrations (2% or more), the baking quality decreased [The other chemical quality factors of the oat flour samples such as protein, starch, total dietary fibre, and ash contents had no correlations with the baking quality parameters. β-glucan content of the oat flours showed only a weak positive correlation with bread crumb hardness, and only after one day of storage  correlated with optimised dough yield . The relation between oat flour particle size and optimal dough yield has not been published before. In gluten-free sorghum baking with optimised water addition, the small particle size of the flours increased the bread specific volume and decreased the bread hardness [p < 0.05), crumb hardness , and staling rate . Thus, small particle size was related to the higher water absorption of the dough, higher water evaporation during baking, lower bread hardness, and lower staling rate. Although the oat flour samples of our study were milled in similar conditions, the particle sizes of the samples varied and caused variation in the oat baking quality, as well. However, we could not identify the cause of the particle size variation in this study, and thus, further research is needed to understand better how to control the quality of the whole grain oat baking.Among the physicochemical quality factors of the oat flour samples, only the median particle size (Dhardness . In our In our study, it was observed that dough yield optimisation was an important quality factor in whole grain oat baking, as most of the oat cultivar samples were baked with good quality by dough yield optimisation. However, variation was observed in the baking quality of twenty oat cultivar samples, as all samples could not be baked with good quality at high dough yields. In whole grain oat baking at optimised dough yield, high fat content and small median particle size of the oat flours had a significant positive influence on the baking quality. This influence needs to be studied in more detail to understand the baking quality of whole grain oat at a deeper level. However, the results of this study can already help millers and bakers to optimise and develop whole grain oat baking in the industrial level processes.