Different ageing technology of wine spirits (WSs) has been investigated, but little has been published on the chemical evolution of aged WS during storage in bottle. The purpose of this study was to examine how 12 months of storage in bottle affected the evolution of antioxidant activity , total phenolic index (TPI) and low molecular weight (LMW) compounds content of the WSs aged through alternative technology using three micro-oxygenation levels (MOX) and nitrogen control (N). Results revealed the ability of phenolic compounds from aged WSs to scavenge free radicals during storage in bottle. Among the in vitro antioxidant-activity methods, FRAP assay was the more effective to differentiate WSs according to the ageing technology. Concerning the overall influence of storage in bottle on antioxidant activity, and TPI and LMW compounds content, the higher results were obtained for the MOX modalities , which showed a similar evolution. In summary, this study provides innovative information, demonstrating that the differences between the aged WSs imparted throughout the ageing process (resulting from different MOX levels) were mostly retained, and only slight modifications during storage in bottle were found.  The alternative ageing technology using wood fragments  combined with micro-oxygenation (MOX) applied to beverages stored in stainless steel tanks intends to simulate the ageing process that occurs in wooden barrels, but in a more sustainable way–less time, lower cost and lower environmental impact ,2. StudiAmong the bioactive compounds, phenolic compounds are one of the most important and well-known group of natural antioxidants. In beverages, the antioxidant activity is mainly correlated with phenolic composition ,9. The aQuercus robur L.) wood, but in particular cases the chestnut (Castanea sativa Mill) wood has gained prominence due to sustainable aspects [In addition, the phenolic compounds of the aged WS are almost exclusively extracted from the wood . The woo aspects . Compari aspects . On the  aspects ,22. In a previous study, in which the WS was aged with wood staves (from chestnut and Limousin oak wood) and a single MOX level (2 mg/L/month), positive and significant correlations between ellagic acid, gallic acid, vanillin and syringaldehyde, and the antioxidant activity assessed by the DPPH method were found .https://projects.iniav.pt/oxyrebrand/index.php/pt/ (accessed on 25 November 2021)) by studying the effect of three levels of MOX or nitrogen (control) applied to the same WS kept in 50 L glass demijohns with chestnut wood staves inside. Since the antioxidant activities of the bioactive compounds are affected by their chemical structure, oxidation conditions and concentration [Research is currently in course under the Project Oxyrebrand  or single electron transfer (SET) mechanisms . SpectroIn this project, despite the interesting results attained on some physicochemical and sensory features of the WSs during the ageing process ,6,27, itThe results regarding the method’s donating capacity of electrons (SET) provide important information on their intrinsic antioxidant potential with minimum environmental interference . It is cIn addition, little is known about the changes in the antioxidant activity of aged WS that occur during storage in bottle compared to the initial storage time (end of the ageing process). The statistical analysis revealed that the antioxidant activities of the WS from the four ageing modalities assessed by the DPPH, FRAP and ABTS assays were not significantly different in the beginning of storage (t0). Little difference in antioxidant activity values  between the ageing modalities may be ascribed to the MOX level and the wood variability (even with the same surface to volume ratio used). After six months in bottle (t6), an increase in the antioxidant activities by DPPH, FRAP and ABTS assays for the WSs resulting from the four ageing modalities was observed. This increase was significantly higher in O15 and O30 by DPPH assay, in O60 and N by FRAP assay, and in all modalities by ABTS assay. In addition, the O15 WS exhibited significantly higher antioxidant activity by DPPH assay than the WSs from other ageing modalities, whereas the O30 had higher antioxidant activity by FRAP assay. These changes in the antioxidant activities are presumably due to differential transformation of phenolic compounds into polymeric and condensed forms, under different oxygen content resulting from the MOX levels combined with the action of factors ruling the storage in bottle . Indeed, these compounds exhibit different chemical properties and reactivity towards the organic radical reagent (DPPH and ABTS assays) and with a Fe(III) complex (FRAP assay) , accordiOn the other hand, at t6 the FRAP assay showed higher antioxidant activity values than DPPH and ABTS assays for the studied WSs, whereas the last ones displayed similar values. These results are in accordance with those of Riviero-Pérez et al.  for red After 12 months in the bottle (t12), the antioxidant activity by the DPPH and FRAP assays decreased significantly only in O15 WS and control WS, respectively. On the contrary, the antioxidant activity of O15 WS increased significantly by ABTS assay. Comparing the WS from the four ageing modalities at this storage time, significantly lower antioxidant activity  in the control (N) was observed, while the MOX modalities behaved similarly. The oxidation and polymerization reactions of LMW compounds during storage may lead to changes in the antioxidant activity of the aged WS, as a consequence of changes in the chemical composition mentioned above ,37 and rRegarding the average results of the three assays performed, DPPH assay showed the lowest values at t12. This behaviour can be ascribed to the reaction mechanism implied in the DPPH assay, in which many antioxidants may react with different kinetics or may not react at all . FurtherConsidering all results, the current study suggests that the FRAP assay was the more effective method for differentiating WSs, based on the ageing modality, during storage in bottle. It also reveals the efficiency of phenolic compounds from the aged WSs to scavenge free radicals during storage in bottle, suggesting that these bioactive compounds are preserved after the ageing process  and may have positive health benefits for the consumers. Similar results were reported in previous studies using the same in vitro methods, demonstrating that the antioxidant activity of white and red wines increased during storage in bottle ,38. The antioxidant activity of the aged WS, which has been clearly demonstrated in several studies, is mostly assigned to its phenolic composition ,23,42. FTPI and LMW compounds contents were not significantly different between the WSs from the four modalities at t0. Therefore, the different MOX flow rates applied during the ageing process with chestnut wood staves did not promote significant differences in the phenolic content between the ageing modalities , as observed at the end of the ageing process . These rpara position, electron-donors at both ortho positions and stabilization via two intermolecular hydrogen bonds with ortho hydroxyl groups [Regarding the evolution over the storage time, the total LMW compounds content did not change significantly regardless of the ageing modality . The TPI of the aged WSs decreased after 6 months of storage in bottle for all ageing modalities, showing significant differences for O30, O60 and N, and then increased up to 12 months. Moganas et al.  reportedl groups . Indeed,l groups ,50. Thisl groups  also repThe slight changes observed in the LMW compounds and antioxidant activities from 6 to 12 months of storage in bottle may be ascribed to the lower level of available oxygen (uptake from headspace and/or dissolved into the aged WS). However, the significant increase of the TPI values found for all WSs may derive from the hydrolysis of phenolic glycosides and hydrolyzed tannins giving rise to free forms, causing this effect ,50. It should be noted that in each storage time, the TPI and total LMW compounds content of the control WS (N) were slightly lower than the those of WS from MOX modalities , which is in line with the pattern observed throughout the ageing experiment . These fΣDPPH = 0.88; rΣFRAP = 0.94 and rΣABTS = 0.89. Furthermore, total LMW compounds also showed a significant correlation with values of the antioxidant activity by FRAP assay (r ΣFRAP = 0.77) and DPPH assay (rΣDPPH = 0.72); however, no correlation was obtained with the ABTS assay. The higher correlations found between TPI, DPPH, FRAP and ABTS assays suggest that synergy between different phenolic compounds  determine the antioxidant activity of the aged WS, as reported by Nocera et al. [In summary, the results of antioxidant activity evolution over time were coherent with those of phenolic composition obtained for the ageing modalities. According to the correlation analysis, a positive and significant correlation between the results of the antioxidant activity determined by three methods and TPI exist: ra et al. .The evolution of LMW compounds was assessed individually over the storage in bottle . These cAmong the quantified LMW phenolic acids, gallic acid, followed by ellagic acid, was the most abundant in the aged WS from the four modalities, whereas ferulic acid had the lowest concentration . Gallic Concerning ellagic acid in aged WS, the ellagitannin degradation during the heat treatment of the wood staves and ellagitannin hydrolysis during the ageing process are the major sources . Also, vRegarding the phenolic aldehydes, they are mainly produced by thermal degradation of the terminal monomer units of lignin, and the cinammic aldehydes can also give rise to benzoic aldehydes during the heat treatment in cooperage ,57. In aThus, the storage in bottle had low impact on the aged WSs phenolic profile. The low concentrations exhibited by the WS from control modality (N) are in accordance with the results attained at the end of the ageing process , confirmRegarding the general effect of storage in bottle on the antioxidant activity, TPI and LMW compounds content of the aged WSs, similar evolution of the WSs from the MOX modalities  was observed. In addition, differences between the aged WSs imparted during the ageing process (resulting from different MOX levels) were mostly preserved. Principal Component Analysis (PCA) was used to study the overall effect of the storage in bottle on the aged WSs’ characteristics  and methanol  were purchased from Merck , and ethanol  was purchased from Carlo Erba . The ultrapure water (conductivity < 0.055 µS/cm) and distilled water (conductivity < 6.0 µS/cm) were obtained from Arium Comfort System . Trolox , TPTZ -s-triazine), ABTS  and sodium acetate trihydrate were purchased from Sigma-Aldrich . DPPH  radical was purchased from TCI . Potassium persulfate (K2S2O8) and iron(III) chloride hexahydrate (FeCl3·6H2O) were purchased from Honeywell Fluka . Gallic acid monohydrate , ellagic acid dehydrate (ellag), vanillic acid (van), syringic acid (syr), ferulic acid (ferul), furfural (furf), 5-hydroxymethylfurfural (HMF), 5-methylfurfural (5mfurf), and vanillin (vanil) were purchased from Fluka . Syringaldehyde (syrde), coniferaldehyde (cofde), sinapaldehyde (sipde) and 4-hydroxybenzaldehyde were purchased from Sigma-Aldrich . All standard compounds had purity > 97%. The standard solutions were freshly prepared prior to use with ethanol/water . Formic acid (98% The experiment comprised two phases : Castanea sativa Mill.) staves manufactured by J. M. Gonçalves cooperage  with medium plus toasting level were inserted into the demijohns of all modalities and reproducing the surface area to volume ratio of a 250 L barrel. The same wine distillate  produced by Adega Cooperativa da Lourinhã  was used to fill the demijohns. MOX was applied to the WS during the ageing time, providing pure oxygen  through a multiple diffuser micro-oxygenator  with ceramic diffusers, at different flow rates according to the ageing modality (O). In the control modality (N), pure nitrogen  was applied continuously over the ageing time through a specific device  to the WS aiming to decrease the dissolved oxygen as much as possible, thus acting as a control. The eight experimental units were stored in the cellar of Adega Cooperativa da Lourinhã in similar environmental conditions.(1) Ageing trial-was carried out in 50 L glass demijohns , encompassing four ageing modalities: one control modality with nitrogen application, and three MOX modalities , with two replicates. The corresponding experimental design was described in detail by Canas et al. [®, Bemis Company, Neenah, WI, USA) to avoid evaporation. The bottles were transported in the same day and stored in the cellar of INIAV-Dois Portos at 19 °C and 80% relative humidity for 12 months. Sampling was carried out at 0, 6 and 12 months. A total of 48 samples  of WS were taken and analyzed to determine the antioxidant activities, total phenolic index and low molecular weight composition, and their correlations as well.(2) Storage in bottle-after 365 days of ageing, the eight aged WS were bottled in the same day in 750 mL amber glass bottles (two bottles from each demijohn), assuring the same level of WS in each bottle. Indeed, the headspace was set at 9.8 mL of air in all bottles to assure that the oxygen ingress into the aged WS was similar, so that the oxidation  promoted−5 M DPPH methanolic solution in a glass tube wrapped with aluminum foil. The tube was vortexed for 10 s and then immediately placed in a water bath  at 30 ± 1 °C for 60 min; the tube was shaken every 10 min and placed back in the water bath. The absorbance was measured at a wavelength of 515 nm using a Varian Cary 100 Bio spectrophotometer . The control absorbance (10 µL de methanol and 3 mL of 8.5 × 10−5 M DPPH methanolic solution) was measured at the beginning and end of the assay. Trolox was used as a reference standard curve (1–15 mM TEAC) and the results were expressed as mmol Trolox equivalent antioxidant capacity (TEAC)/L of WS. The analyses were performed in triplicate.The DPPH  free radical scavenging activity was performed using the modified method described by Nocera et al. . Briefly•+ radical cations were prepared by reacting 4 mL of a 7 mmol/L ABTS stock solution with 70.4 μL of 140 mmol/L potassium persulfate solution for 16 h, at room temperature and in the dark. Thereafter, the ABTS solution was diluted by adding ethanol (99.5%) to the ABTS•+ radical solution until measured absorbance reached 0.700 ± 0.02 at 734 nm using a Varian Cary 100 Bio spectrophotometer . The WSs were diluted with absolute ethanol (1:50 v/v). 3 mL of ABTS solution was added to 30 µL of sample or 30 µL of standard solution, mixed and placed in the water bath  for 6 min at 30 °C. After reaction, the absorbance was measured at 734 nm at room temperature. The control absorbance (30 µL of absolute ethanol plus 3 mL of ABTS solution) was measured at the beginning and end of the assay. Trolox standard curve (0.08–2.0 mM TEAC) was used as reference antioxidant. Results were expressed as mmol Trolox equivalent antioxidant capacity (TEAC)/L of WS. The analysis was carried out in triplicate.The ABTS assay was based on the method reported by Rufino et al.  with som3·6H2O in the dark. The WSs were diluted with absolute ethanol . Sample or standard solutions, distilled water and FRAP reagent  were mixed and kept in a water bath  for 30 min at 37 °C. After cooling to room temperature, absorbance was measured at 595 nm using a Varian Cary 100 Bio spectrophotometer . The control absorbance  was measured at the beginning of the assay. A Trolox standard curve was also prepared (0.08–1.5 mM TEAC). Results were expressed as mmol Trolox equivalent antioxidant capacity (TEAC)/L of WS. The assay was performed in triplicate.The ferric reducing ability was determined by the FRAP (Ferric Reducing Antioxidant Power) method, with modifications ,64. The The study of linearity of Trolox standard curves used in the antioxidant activity assays  was performed using Minitab version 14  and showed that the linear regression was the best model for establishing a relationship between the percentage of DPPH inhibition/or ABTS inhibition/or FRAP reduction) and Trolox concentration (mM) (data not shown).The total phenolic index (TPI) of the WSs was determined according to Cetó et al. . Brieflyv/v) as eluent A, and methanol/water/formic acid (70:28:2 v/v/v) as eluent B, at a flow rate of 1 mL/min and column temperature of 40 °C. All solvents were filtered through a 0.45 µm PVDF membrane  prior to analysis. The auto sampler’s temperature was set at 18 °C and the injection volume was 20 µL. Samples were spiked with an internal standard . The elution program was as follows: 0–3 min at 0% isocratic B; 3–25 min from 0% to 40% B; 25–43 min from 40% to 60% B; 43–55 min at 60% isocratic B; 55–60 min from 60 to 80% A; 60–65 min at 80% isocratic B; 65–75 min from 80 to 0% B, and finally returning to the initial conditions. Detection was made at 280 nm for phenolic acids  and furanic aldehydes , and at 320 nm for phenolic aldehydes . Quantification of these compounds was performed through calibration curves (mg/L).Low molecular weight (LMW) compounds of aged WSs were quantified according to the method of Canas et al. . Chromatp < 0.05) was found. The correlation between antioxidant activities, and TPI and LMW compound concentrations were determined through the Pearson’s correlation coefficient test, considering a confidence level of 95% (p < 0.05). Principal component analysis (PCA) of results was used to evaluate the possible grouping of total LWS compounds for four modalities during the storage time in bottle. Statistical analysis and PCA were performed using Statistica version 7.0 . The results of scores and loadings are standardized and present in the same graphic that identifies the influence of each factor.Data of antioxidant activities, TPI and low molecular weight composition were expressed as mean ± standard deviation of independent duplicates. One-way analysis of variance (ANOVA) was applied to assess the effects of the ageing modalities on the antioxidant activities, and the TPI and LMW compound contents of the aged WSs for each storage time in bottle. Another one-way ANOVA was carried out to assess the significance of the antioxidant activities, and the TPI and LMW compounds over storage time in bottle. Tukey’s test was made to compare the average values when a significant difference  had significantly lower antioxidant activity and TPI values. Therefore, the differences between the aged wine spirits imparted by the ageing process (resulting from different oxygenation levels) were mostly retained. Slightly higher performance of the micro-oxygenation modality with higher oxygen application is in accordance with our previous studies, reinforcing that this technological option seems to be the most suitable for wine spirit quality and ageing sustainability.


ESR2) and H19 promoters and their influence on phenotype modifications in a cohort of AFAB people at baseline (T0) and after 6 mo (T6) and 12 mo (T12) of testosterone therapy . A total of 13 AFAB subjects (mean age 29.3 ± 12.6) were recruited. The percentage of methylation of the ESR2 promoter significantly increased at T6 (adj. p = 0.001) and T12 (adj. p = 0.05), while no difference was detected for H19 (p = 0.237). Methylation levels were not associated with androgen receptor (AR)/estrogen receptor beta (ERβ) polymorphisms nor hormone levels at baseline and after six months of treatment. On the other hand, total testosterone level and patient age resulted in being significantly associated with ESR2 methylation after twelve months of treatment. Finally, the difference in ESR2 promoter methylation between T6 and baseline was significantly associated with the number of CA repeats of the ERβ receptor, adjusted vs. all considered variables . No associations were found with CAG repeats of the AR, age, and estradiol and testosterone levels. Despite the small sample size, we can hypothesize that treatment with exogenous testosterone can modify the ESR2 methylation pattern. Our data also indicated that epigenetic changes may be regulated, suggesting that the modulation of estrogen signaling is relevant shortly after the beginning of the treatment up to T6, with no further significant modification at T12. Furthermore, estrogen receptor methylation appears to be associated with the age of the subjects and exogenous testosterone administration, representing a marker of androgenic treatment. Nonetheless, it will be necessary to increase the number of subjects to evaluate how epigenetic regulation might play a relevant role in the modulation of phenotypical changes after testosterone treatment.Virilization of gender-incongruent subjects to whom were assigned the female gender at birth (AFAB) is achieved through testosterone administration. Inter-individual differences in the timing and acquisition of phenotypic characteristics, even if the same hormone preparations and regimens are used, are frequently observed. Polymorphisms of sex hormone receptors and methylation of their gene promoters, as well of several imprinted genes as H19, may underlie the differential response to treatment. Thus, the aim of this study was to examine the possible relationship between the CpG methylation profile of the estrogen receptor 2 gene ( Gender-affirming hormonal therapy (GAHT) is critical for phenotypical and psychological transition in adults with gender incongruence (GI), which is an enduring and marked incongruence between the individual’s desired gender and the assigned sex at birth. While this definition includes a broad spectrum of transgender people (stretching between binary and non-binary subjects), the current scientific literature generally refers to assigned females at birth (AFAB) and assigned males at birth (AMAB) subjects .Transgender people are often considered a psychologically vulnerable group compared to the general population, partly due to distress deriving from GI itself and partly to a non-inclusive cultural background, which might also include difficulties in accessing general healthcare and GAHT ,2. GAHT,This is achieved through the reduction of endogenous sex hormones and their replacement with exogenous hormones, allowing the progressive substitution of the incongruent patient’s secondary sexual characteristics with the desired ones. In virilizing therapy for incongruent subjects who have been assigned the female gender at birth (AFAB people), the treatment of choice relies on one of the many testosterone preparations available, following the principles of hormone replacement therapy in hypogonadal patients . CessatiAR), and a few authors have investigated the occurrence of modifications of the estrogen receptor 1 gene (ESR1) promoter methylation after GAHT [AR and ESR1 genes’ methylation could play a role in modulating the induction of desired phenotypical changes, as well as being a putative marker of hormonal treatment. On the other hand, no data are available on the estrogen receptor 2 (ESR2) gene.Scant data are available for the androgen receptor  [H19 has also been associated with pubic hair onset and genital or breast development in adolescent boys and girls, respectively [H19 promoter methylation could be potentially related to a differential development of secondary sex characteristics after GAHT.Moreover, ectively . Thus, tESR2 and H19 promoters and their possible influence on acquired changes in a cohort of AFAB people at baseline (T0) and after 6 mo (T6) and 12 mo (T12) of testosterone therapy.Thus, the main purpose of this study was to examine the possible relationship between the CpG methylation profile of We prospectively recruited consecutive AFAB people referring to the Gender Incongruence Ambulatory of the Department of Experimental Medicine (AOU Policlinico Umberto I—“Sapienza” University of Rome) before administration of GAHT. The diagnosis of gender dysphoria was confirmed by mental health specialists , according to the DSM-V criteria. All patients provided their informed consent before the start of the treatment and the study was approved by Ethics Committee “Sapienza” . Each patient underwent physical examination, Ferriman–Gallwey scoring , and horEach recruited subject provided a peripheral blood sample (8 a.m.) after overnight fasting. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were quantified by Chemiluminescent Microparticle ImmunoAssay . Detection limits, intra- and inter-assay coefficients of variation, as well as normal ranges from our laboratory were previously described .DNA was extracted from peripheral blood leukocytes using the Wizard Genomic DNA Purification Kit . Extracted DNA was quantified by NanoDrop ND-2000 . For the androgen receptor, located on the X chromosome, the analysis of the percentage of the inactivation of the alleles was performed as proposed by Zitzmann et al. (2004) . BrieflyThe percentage of X chromosome inactivation was then calculated using Gene Mapper Analysis software v. 4.1 . Variables were defined as: digested allele 1 (A), digested allele 2 (B), non-digested allele 1 (C), and non-digested allele 2 (D). Normalization of the results from the two independent reactions and compensation for iniquitous allele amplifications due to confounding factors other than methylation were achieved through a correction factor :Allele 1The extreme values of 0 and 1 correspond to no inactivation and complete inactivation, respectively. A value of 0.5 was interpreted as a random inactivation. The sum of inactivation percentages of alleles 1 and 2 must be equal to 1 (Equation (1) + Equation (2) = 1). To determine the length of the polymorphic fragments in the androgen receptor (AR) and estrogen receptor beta (ERβ) genes , DNA samples were analyzed by linear sequencing  using primers flanking the triplets’ repeat regions. The forward primer for CA and CAG was fluorescently labelled with FAM at 5′ to enable the fragment to be seen during electrophoresis  . In SuppESR2 and H19 promoters was performed as previously described by different authors [One-hundred nanograms of DNA was bisulfite-converted using the EpiTect Plus DNA Bisulfite Kit (Qiagen). PCR amplification of the  authors ,16 Bisulp-value of 0.05 was considered significant. Associations between methylation and the other evaluated parameters were evaluated through linear regressions and generalized linear models. All computations were carried out with the Statistical Package for the Social Sciences (SPSS) 25.0 .Continuous variables are presented as the mean ± SD or the median and IQR, depending on the shape of the distribution curve evaluated by the Kolmogorov–Smirnov test. Categorical variables are presented as counts and percentages. Differences in methylation pattern across samples were calculated by Friedman’s test. A two-tailed p < 0.001)  y) provided a blood sample for epigenetic analysis. The baseline characteristics of the caseload and data on AR/ERβ receptors are shown in < 0.001) .ESR2 increased significantly at T6 (adj. p = 0.001) and T12 (adj. p = 0.05) (H19 promoter methylation (p = 0.237).Post-treatment data from this pilot study showed that the percentage of methylation of  = 0.05) , while nESR2 methylation variation with available genetic and hormone parameters. Baseline methylation levels were not associated with either AR/ERβ genetic polymorphisms or hormone levels  and blood testosterone levels .Linear regressions were performed to evaluate the associations of the e levels . Likewisreatment . On the  of GAHT . MoreoveESR2 promoter methylation between T6 and baseline was significantly associated with the number of CA repeats of the ERβ receptor, adjusted vs. all considered variables    . On the  = 0.53) .AR in transgender women compared to cisgender controls [Gender dysphoria is a relatively rare condition, where the strict link between hormone receptor signaling and exogenous hormone administration is likely to affect clinical outcomes and phenotypical changes. However, the few genetic and epigenetic studies in this setting have focused only on their possible contribution on the development of gender identity/incongruence. A recent metanalysis from D’Andrea et al. (2020) confirmed the presence of a significantly higher number of CAG repeats of controls . CautionSeveral articles support the effect of methylation on imprinted genes with growth, growth-related hormone concentrations, adiposity, and birth weight ,18. DespESR1 gene promoter in cisgender and transgender subjects [Hormone receptor  promoters’ methylation is involved in the development of many diseases, such as atherosclerosis, endometriosis, and cancer ,19. DNA subjects . AlthougAR mRNA expression in blood after both testosterone and estradiol treatment in transgender subjects  [AR mRNA in the blood of transgender men, as well as an increase in the methylation of AR and ESR1 in transgender women and men, respectively, after 12 mo of treatment. Remarkably, these changes were correlated with several anthropometric (waist circumference), metabolic , and hormonal  parameters. This result is particularly important, as it suggests how a fine regulation of the hormone signaling may take place under GAHT. The small sample size of this study does not allow generalizing this observation. Finally, the previously cited study from Fernandez et al. (2020) also analyzed the methylation pattern of ESR1 after 6 mo of hormonal treatment [ESR2 after testosterone treatment of AFAB subjects within a similar time frame (6 mo). Furthermore, these changes appeared to remain constant up to 12 mo. Therefore, it is possible to hypothesize that treatment with exogenous testosterone can modify the pattern of methylation and, possibly, gene expression. However, our data based on a small study population need to be validated on a larger number of subjects in order to establish the role of epigenetic regulation in the modulation of phenotypical changes after GAHT. Nonetheless, our data suggest that epigenetic changes may be strictly regulated by testosterone. In fact, the modulation of estrogen signaling seems relevant within the first six months of treatment, when methylation might work in tandem with CA polymorphisms, as we detected through linear models between baseline and T6, maintaining itself for more prolonged treatments. Age might be involved as it seems to be constantly and positively associated with methylation of the ESR2 promoter during GATH. In fact, it is known that human ageing is associated with genetic, epigenetic, and environmental factors, which may be, in turn, associated with the development of diseases [ESR2 promoter. If confirmed on a larger caseload and longer follow-up, we may hypothesize exploiting the modulation of androgenic and estrogenic signaling for GAHT tailoring in transgender men and women. Another potential application of these data could be to use the methylation pattern as a marker of androgenic treatment. Being exogenous androgens are the main determinant of this epigenetic marker at T12, it is possible to speculate that this change may also be present following other forms of exogenous testosterone administration, including doping. Epigenetic modifications have been investigated in the setting of sports medicine, hypothesizing an association between epigenome and physical performance traits [ESR2, the investigation of these effects in the setting of illegal hormone administration  may be warranted.From a clinical point of view, the main impact from functional variants in hormone receptor signaling is expected to be found both in the timing and amplitude of the phenotypical changes after GAHT. In physiological puberty, hormonal activation is expected to be modulated by the genetic background and the epigenome ,22. Althctively) . More rectively) . The autreatment . The autdiseases . Age-depdiseases ,28, and diseases . Anothere traits . Histonee traits , althougESR2 promoter methylation is increased after testosterone treatment and remains constant up to one year of treatment. The associations we detected with age and testosterone levels suggest that epigenetic modifications might be modulated from these parameters during treatment. Instead, we could not demonstrate a role for H19 promoter methylation, a parentally imprinted gene. Future studies are needed in order to explore the sexually differentiated mechanisms behind DNA methylation considering epigenetic regulation in a larger panel of genes that may directly or indirectly influence hormone treatment-related response.In conclusion, data from this pilot study confirm the safety and efficacy of testosterone treatment in AFAB people. Nonetheless, it is likely that epigenetically modulated hormone signaling may not only affect the timing of phenotype changes, but also may represent a molecular marker of hormone utilization/abuse in both transgender and cisgender people . Current


An investigation of mechanical properties of Ti6Al4V produced by additive manufacturing (AM) in the as-printed condition have been conducted and compared with wrought alloys. The AM samples were built by Selective Laser Melting (SLM) and Electron Beam Melting (EBM) in 0°, 45° and 90°—relative to horizontal direction. Similarly, the wrought samples were also cut and tested in the same directions relative to the plate rolling direction. The microstructures of the samples were significantly different on all samples. α′ martensite was observed on the SLM, acicular α on EBM and combination of both on the wrought alloy. EBM samples had higher surface roughness (Ra) compared with both SLM and wrought alloy. SLM samples were comparatively harder than wrought alloy and EBM. Tensile strength of the wrought alloy was higher in all directions except for 45°, where SLM samples showed higher strength than both EBM and wrought alloy on that direction. The ductility of the wrought alloy was consistently higher than both SLM and EBM indicated by clear necking feature on the wrought alloy samples. Dimples were observed on all fracture surfaces.  Additive manufacturing (AM) has gained huge popularity in the last decade or so. Its ability to produce complex shapes, reduce the number of parts, reduce the material waste, etc. have been the main drive of this technology. According to ASTM, AM can be devided into seven categories including (i) VAT photopolymerisation, (ii) material jetting, (iii) binder jetting, (iv) sheet lamination, (v) material extrusion, (vi) powder bed fusion (PBF), and (vii) directed energy deposition (DED). For metallic materials, Directed Energy Deposition (DED) and Powder Bed Fusion (PBF) are the only options. Within the PBF methods, electron beam melting (EBM) and selective laser melting (SLM) are two common methods to produce metallic parts where the powder is melted and fused by either electron beam or laser beam. SLM materal is generally stronger and less ductile compared with EBM . SLM proAs far as the metallic material is concerned, some of the metals/alloys which are readily used for AM including stainless steels (SS316 and SS304), nickel-based alloys Inconel 718/625), aluminium alloys , Co-Cr alloy, tool steels (H13), and titanium (Ti6Al4V and CP Ti). The above metallic AM has been reviewed by a few authors 18/625, a.More specifically, evaluation of mechanical properties of AM of Ti6Al4V produced by EBM and SLM have been reported separately by various authors ,19,20,21EBM and SLM are the two most common methods to produce metallic componets, therefore, this investigation was dedicated to focus on the Ti6Al4V samples manufactured by EBM and SLM, and compared to the traditional wrought alloys. To the best knowledge of the authors, there has been no publications on a comprehensive comparison between EBM, SLM and wrought alloys comparing various build directions, hence, the novelty of this paper. It presents a comparison of surface roughness, microsctructures and mechanical properties of samples manufactured by EBM and SLM built in the 0°, 45° and 90° in the as-printed conditions, compared with those of wrought alloys  also in three different directions. The material investigated in this study was Ti-6Al-4V (Ti64) manufactured by three different processes, i.e., (i) wrought and rolled sheet, hereafter wrought (ii) electron beam melting-EBM, and (iii) selective laser melting-SLM with a thickness of around 2 mm . For the3. The SLM samples were printed using EOS GmbH, EOSINT M270 machine using a rotating stripe pattern. Parameters of 190 W laser power, scanning velocity of 1200 mm/s, hatch distance of 100 μm, and layer thickness of around 30 μm were used, resulting in a calculated energy density of 53 J/mm3.The EBM samples were printed using Arcam Q10plus 5.0 themes with a layer thickness of 50 μm, speed function of 64, line offset of 0.2 mm and a current of 30 mA which would give an energy density of around 94 J/mmThe densities of the printed samples were measured by the Archimedes methods.Surface roughness, Ra, was measured according to layer direction using Taylor Hobson Ultra 2006 version software. An average of three readings was taken per sample.3 + 100 mL water). An optical microcope , was employed to observed the microctructure. The microscope is equipped with an image analysis . A scanning electron microscope  was employed to examine the surface morphology of the as-printed samples and the fracture surfaces of the tensile tested samples.Cross-sectional metallography samples were prepared from each material. The samples were ground were ground using SiC papers with grit size from 100 to 2400, and were polished up to 0.3 μm diamond paste. A diluted hydrogen peroxide (30%) was used at the final stage of polishing for a better response to ecthing. The samples were then etched with Kroll’s reagent  being the weakest and least ductile. This is in agreement with what was reported by Ren et al. [n et al.  and Agiun et al. . It is bn et al. ,13). Forn et al.  showed sn et al. ,37,38,39With regards to the stiffness, the slope in the elastic region on the stress vs. displacement graph showed that SLM had a slightly higher stiffness than both EBM and wrought samples in all three orientations .The results from tensile testing represented by the digital image corelation (DIC) analysis showed a clear indication that wrought materials had more ductility compared with AM materials . EBM samFrom the DIC images it is also obvious that the highest strain took place in the reduced sections area, hence, fracture locations. The local strain where fracture took place are indicated in red, and it showed somewhat higher than the total elongation. While the AM samples showed negligible necking , the wroMacrographs of the fracture surfaces from tensile test samples are shown in The fracture surfaces of all samples showed dimples  resultinDimples on the wrought samples were more regular and their distribution was also more uniform. The SLM samples had some small spherical gas pores, while the EBM samples showed some relatively larger irregular (crack-like) pores. From the shape of the pores, it can be assumed that the cause of those pores were different, e.g., gas entrapment around the melt pool on the former , and incThe microstructures of the samples were significantly different with α’ martensite on the SLM, acicular α on EBM and combination of both on the wrought alloy.EBM samples had higher surface roughness (Ra) compared with both SLM and wrought alloy. For AM materials, the surface roughness was associated with the size of the particle and the improper melting.SLM samples were comparatively harder than wrought alloy and EBM.Tensile strength of the wrought alloy was higher in all directions except for 45°, where the SLM samples showed higher strength than both EBM and wrought alloy for the same direction. The ductility of the wrought alloy was consistently higher than both SLM and EBM indicated by clear necking feature on the wrought alloy samples. The variations in mechanical properties is believed to be associated with anisotropy on the samples.All the fracture surfaces showed some dimples indicating with wrought samples had more uniform dimples than the AM samples. Defects, i.e., porosity and crack-like defects were present on both SLM and EBM samples.A comparison of Ti6Al4V manufactured by SLM and EBM with wrought alloy has been investigated. Both the SLM and EBM were in the as-printed condition, while the wrought alloy was in the as-received condition. The summary from this investigation are as follows:


Anilany helenae is a Critically Endangered frog native to the central highlands of Madagascar. Due to ongoing habitat loss of its known range, this species’ population is considered declining, while little is known about its ecology, behavior, and taxonomy. Within the context of developing tools that can aid the conservation of Madagascar’s amphibian fauna, and add to the continued understanding of their taxonomy, we assembled its complete mitochondrial genome (Genbank Accession number MZ751042). This contributes the first complete mitochondrial genome of a microhylid from Madagascar, despite there being over 100 species in the Cophylinae subfamily alone. Anilany helenae’s circular mitochondrial genome is 17,519 bp long, contains 37 genes, and exhibits differences in gene arrangement compared with other microhylids, including the placement of protein coding genes nad1 and nad2. A phylogeny of the 13 protein coding genes of the few Madagascan anuran mitogenomes available, along with species from Africa and East Asia, places A. helenae along with the New Guinean Mantophryne lateralis in a basal position with respect to the other microhylids in the tree. Anilany helenae Vallan A. helenae was found in multiple forest fragments within Ambohitantely  as well as 10 km away in Ankafobe, in a small threatened forest fragment . Due to frequent discovery and description of novel species, the taxonomy of Madagascar’s Cophylinae subfamily is continuously debated and revised. Here we describe the complete mitogenome of A. helenae, contributing to the continued research into Madagascar’s anuran taxonomy.This species was originally described as being part of the z et al.  proposedde novo NOVOPlasty organelle assembler and heteroplasmy caller  was collected by Katherine Mullin from the main forest block in Ambohitantely on 13 April 2019 at −18.19694, 47.28413 during visual encounter surveys. The specimen was found in the leaf litter on the forest floor. This specimen and the DNA has been deposited in the archives of the Molecular Ecology Lab, Cardiff University. Genomic DNA was extracted from a tissue sample using the QIAGEN DNeasy Blood & Tissue Kit protocol. Whole genome paired-end Illumina sequencing (150 PE) was performed by Novogene, Hong Kong. The raw reads were assembled using the The mitogenome had a GC content of 41.1%, with individual base percentages of: G − 12.9%, C − 28.2%, A − 30.3%, T − 28.5%. The MITOS WebServer  to trnN(aac), which is consistent with the region of genomic reorganization known for seven mantellid genera from Madagascar , NADH dehydrogenase subunit 2 (nad2) and the 16S rRNA genes show rearrangement, with nad2 placed after cob, and before (rather than after) 16S and nad1. Further sequencing with longer reads may be required to fully resolve the gene order and any duplications.The order and orientation of genes show variation when compared with the anurans in our phylogenetic tree . SimilarA. helenae) and 1 hylidae species were used to infer phylogenetic relationships , and that this phylogeny should be reexamined with increased taxon sampling, including additional Madagascar Cophylinae species such as close relatives in the Stumpffia and Anodonthyla genera. Our results support the ongoing taxonomic work on Madagascar’s amphibian fauna.A total of 11 microhylidae (7 from East Asia, 1 from mainland Africa and 2 from Madagascar, in addition to ionships . The 13 ionships , to a to


Noninvasive prenatal testing (NIPT) is the most recent modality widely used in prenatal diagnostics. Commercially available NIPT has high sensitivity and specificity for the common fetal chromosomal aneuploidies. As future advancements in NIPT sequencing technology are becoming promising and more reliable, the ability to detect beyond aneuploidies and to expand detection of submicroscopic genomic alterations, as well as single-gene disorders might become possible.Here we present a case of a 34-year-old pregnant woman, G2P1, who had NIPT screening which detected a terminal microduplication of 10.34 Mb on the long arm of chromosome 15 (15q26.1q26.3). Subsequent prenatal diagnostic testing including karyotype, microarray and fluorescence in situ hybridization (FISH) analyses were performed. Microarray testing confirmed and particularized a copy number gain of 10.66 Mb of the distal end of the long arm of chromosome 15. The G-banding cytogenetic studies yielded results consistent with unbalanced translocation between chromosome 15 and 18. To further characterize the abnormality involving the long arm of chromosome 18 and to map the genomic location of the duplicated 15q more precisely, FISH analysis using specific sub-telomeric probes was performed. FISH analysis confirmed that the extra duplicated segment of chromosome 15 is translocated onto the distal end of the long arm of chromosome 18 at band 18q23. Parental karyotype and FISH studies were performed to see if this unbalanced rearrangement was inherited from a healthy balanced translocation carrier versus being a de novo finding. Parental chromosomal analysis provided no evidence of a rearrangement between chromosome 15 and chromosome 18. The final fetal karyotype was reported as 46,XX,der(18)tdn.In this case study, the microduplication of fetal chromosome 15q26.1q26.3 was accurately detected using NIPT. Our results suggest that further refinements in NIPT have the potential to evolve to a powerful and efficient screening method, which might be used to detect a broad range of chromosomal imbalances. Since microduplications and microdeletions are a potential reportable result with NIPT, this must be included in pre-test counseling. Prenatal diagnostic testing of such findings is strongly recommended. The use of maternal serum marker screening and ultrasound imaging (ultrasonography) to detect chromosome aneuploidies and other birth defects are traditional approaches of prenatal care in the first and/or second trimesters . If thesClinical translation of NIPT technologies has revolutionized prenatal care. NIPT measures and evaluates small fragments of placental DNA that are circulating in a pregnant woman’s blood. The cell-free DNA (cfDNA) in maternal plasma reflects the genetic makeup of the developing fetus. Commercially available NIPT is used as a prenatal screening technique allowing for detection of the most common autosomal and sex chromosome aneuploidies. Further advances in NIPT technology have shown that micropulications and microdeletions can be detected. Many professional societies currently recommend that NIPT be used as a screening tool, not a definitive diagnostic test; therefore, when cell-free DNA results are at high risk, the diagnosis requires subsequent follow-up testing by means of genetic analysis of samples collected invasively. NIPT provides high sensitivity (true positive rate) and specificity (true negative rate), making it an attractive alternative to the serum screens and invasive diagnostics currently in use , 5.A normal human cell is made up of 46 chromosomes that are grouped into 24 different types and arranged in 23 homologous pairs. Of those 23 pairs, 22 are autosomes, and the remaining pair is comprised of the two different types of two sex chromosomes, which specify gender  . ChromosHerein, we present a clinical prenatal case in which NIPT testing detected a 10.34 Mb gain (duplication) of the long arm of chromosome 15. Clinical value of NIPT finding was further confirmed by microarray analysis in conjunction with karyotyping and FISH analysis.A 34-year-old pregnant woman, G2P1, gestational age 10 weeks and 5 days, came to prenatal clinic for routine prenatal care. Patient elected to screen for the common fetal chromosomal aneuploidies using NIFTY standard panel. Routine first trimester ultrasound examination did not indicate any fetal abnormalities. No history of spontaneous abortions was reported. Family history was remarkable, no chromosomal anomalies or other genetic disorders were reported.Noninvasive screening test was performed at NIFTY . A blood sample required (minimum of 10 ml) for NIPT testing was drawn at > 10 weeks of gestation. NIFTY standard panel screens for the common autosomal aneuploidies and sex chromosome aneuploidies.Genomic microarray analysis on isolated DNA from amniotic fluid cells was performed at Life Code Laboratories . Briefly, genomic DNA from amniotic fluid was extracted using NucleoSpin blood extraction kit by Macherey–Nagel. CytoScan® Optima Array by Affymetrix  was used for the detection of copy number variations (CNV) and loss of heterozygosity (LOH), according to manufacturer’s instructions. Results were analyzed using Chromosome Analysis Suite (ChAS). Additional databases referenced for the analysis included: Decipher, DGV, ClinVar, OMIM, NetAffyx, UCSC, and Ensemble.Cytogenetic analysis on long-term cultured amniocytes was performed using standard cytogenetic techniques according to specimen specific protocols, in accordance with the European Society of Human Genetics (ESHG) and European Cytogenetics Association (E.C.A) guidelines. Chromosomes were aged and banded using G-bands by pancreatin and Giemsa staining technique. To investigate the total number and structure of the chromosomes, twenty metaphase cells were visualized and analyzed by qualified cytogenetics technologist using Zeiss microscope Axioskop2 plus  with the assistance of the Metasystems imaging system. Furthermore, to rule out a balanced chromosomal rearrangement, involving chromosome 15, parental chromosome analysis from a stimulated peripheral blood lymphocytes was performed.Fluorescence In Situ Hybridization (FISH) was performed at the Klinikum rechts der Isar laboratories . FISH analysis was performed using commercial Vysis sub-telomeric probes for chromosome 15  and 18 . All specimen types were subjected to standard FISH pretreatment, hybridization, and fluorescence microscopy according to the manufacturer’s specifications and standard specimen specific laboratory protocols. The results of genetic testing were described and reported in accordance with the International Standing Committee on Human Cytogenetic Nomenclature .Professional and detailed genetic counseling regarding the NIPT screening, purpose, significance, accuracy, limitations, and other screening and diagnostic testing options was provided. Informed consent for genetic testing was obtained. A duplication of 10.34 Mb of the long arm of chromosome 15, specifically 15q26.1-q26.3 was reported by noninvasive prenatal testing as an incidental finding. Reported fetal cfDNA was 10.66%. Amniocentesis was performed under the guidance of ultrasound, where approximately 18 ml of amniotic fluid was withdrawn by syringe for diagnostic studies.The results of the chromosomal microarray analysis confirmed the presence of a pathogenic 10.66 Mb gain of the distal end of chromosome 15, chromosomal region 15q26.1q26.3. More specifically, the duplicated segment includes the chromosomal region between bases 91,763,147 and 102,429,112, encompassing 22 OMIM genes (NCBI Build 37/hg19)  and 18 . Following hybridization, the probe signal pattern noted was three signals  for chromosome 15q and two signals for chromosome 18q. Two green signals localized to anticipated chromosome 15q region, the third signal being localized to structurally abnormal chromosome 18q, resulting in an unbalanced rearrangement indicated by a single fusion signal Fig. B.Studies for sub-telomeric region of the long arm of chromosome 18, showed two probe signals and the probes were localized to their anticipated 18q sub-telomeric bands. Thus, the FISH studies provided no evidence for a rearrangement or loss of the sub-telomeric region of chromosome 18. The FISH test confirmed the characterization of the abnormalities involving chromosomes 15 and 18 that were noted in both the microarray and G-banding analyses.No structural or numerical chromosomal anomalies were detected in parental chromosome analysis Fig.  A and B.Parental metaphase FISH analysis showed the expected number of probes  present, and the probes were localized to their anticipated sub-telomeric bands. There was no evidence for loss or gain of the p or q subtelomeric regions for the chromosomal regions evaluated. Thus, parental FISH studies provided no evidence for a rearrangement between chromosome 15 and chromosome 18 Fig.  C and D.Because of improvements in the safety of invasive procedures and advances in technology, the most recent systematic reviews demonstrated pregnancy loss rates for CVS and amniocentesis to be less than 1.0% , 12. NatA concern is that as higher proportions of the genome are analyzed, false positive and false negative results are expected to increase, which would result in an increase in unnecessary invasive procedures . NIPT isPartial trisomy of the distal 15q is a rare chromosomal disorder. In general, the duplication of 15q has been characterized by prenatal and postnatal overgrowth, craniosynostosis, distinct facial features, and intellectual disability, likely reflecting triplosensitivity for one or more of the several genes that are found within this region –20. The Previous study has detected the deletion of 5 Mb in fetal chromosome 15q11.2q13.1 and was further confirmed by CNV and karyotype analysis . Our stuWithout molecular cytogenetic testing modalities, a dup(15)(q26.1q26.3) would have been suggested, but a derivative chromosome 18 would not have been detected. CNV analyses provide information whether copy number gains and losses are present but not whether they have been translocated from their normal position(s) in the genome. Thus, confirmation of suspected chromosome abnormalities by FISH and chromosome analysis may be necessary to determine the nature of an abnormality.The pregnancy reported in this paper ended in an elective interruption of pregnancy. Since unbalanced translocations can result from either de novo event or the malsegregation of a balanced parental translocation, parental chromosomal studies were completed to see if either of them is a balanced carrier of a translocation involving chromosome 15 and 18. Due to lack of precise and targeted genomic testing done in Bosnia and Herzegovina, diagnostic follow up testing was done in collaboration with laboratories in Greece and Germany. Parental karyotype and FISH analysis were normal with no genetic aberrations. The couple has another healthy child with unknown karyotype. The de novo origin of the 15q duplication is consistent with the fact that there is no prior history of spontaneous abortions and no family history of chromosomal anomalies or genetic disorders. Moreover, it is important to note that negative results in both parents cannot exclude the possibility of parental gonadal mosaicism, thus leading to minor recurrence risk in subsequent pregnancies.In conclusion, this case report shows that the resolution of current NIPT technology has the potential to detect submicroscopic aberrations. The clinical application of NIPT screening can be second best choice when patients refuse invasive diagnostic testing, especially for those with no ultrasound findings and no family history. It is clear that NIPT can potentially detect clinically significant unbalanced chromosomal abnormalities present in approximately 1.7% of all structurally normal pregnancies . There i


Domestic turkeys have been bred to reach a body mass of up to three times that of wild turkeys. Most of this increase is from larger muscles, but it is unclear exactly how the components of each muscle have been altered and what that may mean for muscle function. In this study, we looked for potential differences between wild and domestic turkeys in (1) the size of the individual muscle fibers and (2) the collagenous skeleton that supports those fibers in the lateral gastrocnemius muscle, an ankle extensor important for walking. We also measured the force this muscle could produce. The domestic turkey muscle had a greater number of smaller muscle fibers than the wild turkey. The amount of collagen in the domestic turkey muscle was also lower than wild turkeys, likely contributing to meat tenderness. While the domestic turkey lateral gastrocnemius muscles could produce the same amount of force per a given area of muscle, they could only produce half as much force per unit body mass. Selection for increased muscle mass has altered the structure of the lateral gastrocnemius muscle; however, overall body mass likely contributes more to hind limb functional differences observed in the domestic turkey.2) than those of the wild turkey , indicating that the absolutely larger domestic turkey muscles are a result of an increased number of smaller fibers. Collagen concentration in domestic turkey muscle (4.19 ± 1.58 μg hydroxyproline/mg muscle) was significantly lower than in the wild turkeys , with visible differences in endomysium texture, observed via scanning electron microscopy. Selection for increased muscle mass has altered the structure of the LG muscle; however, scaling likely contributes more to hind limb functional differences observed in the domestic turkey.Selection for increased muscle mass in domestic turkeys has resulted in muscles twice the size of those found in wild turkeys. This study characterizes muscle structural changes as well as functional differences in muscle performance associated with selection for increased muscle mass. We compared peak isometric force production, whole muscle and individual fiber cross-sectional area (CSA), connective tissue collagen concentration and structure of the lateral gastrocnemius (LG) muscle in wild and adult domestic turkeys. We also explored changes with age between juvenile and adult domestic turkeys. We found that the domestic turkey’s LG muscle can produce the same force per cross-sectional area as a wild turkey; however, due to scaling, domestic adults produce less force per unit body mass. Domestic turkey muscle fibers were slightly smaller in CSA (3802 ± 2223 μm The domestic turkey has undergone major morphological changes due to artificial selection for high growth rates and increased muscle mass, as well as advances in nutrition that have led to an increase in body mass . This haDomestic turkeys experience locomotor issues due to genetics, growth rate and body conformation, among other factors ,7,8. SteSkeletal muscle is made up of both contractile and connective tissue elements, and changes to either component can affect muscle function ,14,15. MSkeletal muscle structural changes are important to understand not only for animal health and welfare, but also for meat quality purposes. The proportions of contractile and connective tissue components affect meat texture. For example, increased connective tissue makes cooked meat tougher and less tender . UnderstIn this study, we investigate structural and functional differences in the lateral gastrocnemius muscles of domestic and wild turkeys. We also explore how domestic turkey muscle changes structurally and mechanically during ontogeny to better understand how increased muscle mass may affect locomotor performance at different life stages. We use adult wild turkey muscle as a control group for optimal mechanical performance in this species. The lateral gastrocnemius functions as an ankle extensor crucial to normal locomotion in turkeys, and extensive work characterizing this muscle’s mechanical properties and in vivo performance in wild turkeys is available ,24,25,26Meleagris gallopavo, were obtained from local farms between 2013 and 2016. Turkeys were either housed and raised in the Animal Care facilities at Brown University or euthanized immediately upon arrival. Six juvenile domestic turkeys raised on pasture were obtained between 8–12 weeks in age from a local farm. Collagen samples, microscopy samples and force measurements were taken upon arrival to the laboratory. Adult turkeys were all over 18 weeks old when experiments were performed. Ages of data collection for muscle collagen concentrations and SEM image analysis can be found in the n = 6 in 2013 and n = 4 in 2016). Muscle force data was collected from all ten, while collagen samples were only taken from the four individuals in 2016. Broad breasted white turkeys, Wild turkeys were also kept in the animal care facilities, with some obtained 3 days post-hatch and others obtained as adults post-breeding. Muscle samples for collagen measurements and imaging were taken from four adult wild turkeys from the 2015 cohort raised in our facilities. Force data were collected from 20 total wild turkeys in conjunction with other ongoing studies within the lab. For ten of the twenty wild individuals, force measurements were obtained between the ages of 18 weeks and just over one year old. For the remaining ten, force measurements were obtained during previous data collections from birds >3 years old. Please note, wild turkeys are much longer lived than domestic turkeys, so wild turkeys could be sampled over a wider age range.2 in area, with a minimum of 15 ft2 per individual. The floor was concrete with ample wood shavings, environmental enrichment devices and structures for roosting. The Brown University Institutional Care and Use Committee approved all animal use, IACUC no. 1602000189, and complied with state and federal legislation and regulation. Individual bird information can be found in the All turkeys were maintained on an ad libitum water and 28% protein commercial poultry diet for the first 8 weeks and then transitioned to 21% protein poultry feed. Birds raised on pasture likely also ate insects in their environment. Oyster shell grit was provided for calcium, and all birds received mealworm treats during concurrent locomotion studies. The flock lived in an open pen system approximately 300 ft0) for each individual turkey. Animals were euthanized at the completion of measurements.The surgical and experimental procedure for measuring peak isometric muscle force closely followed previous methods ,27,28. Tm is the mass of the muscle in g, θ is the pennation angle in degrees, ρ is density of 1.06 g/cm3 [fL is muscle fascicle length in cm. Wild turkey data were taken from previous wild turkey experiments in the lab, including [The physiological cross-sectional area of the muscle was calculated with the equation:06 g/cm3  and Lf including , and froVertebrate skeletal muscle force is reasonably uniform in proportion to fiber cross-sectional area, ranging from 150 to 300 kPa, with an average of 200 kPa . A predi2O for every 10 mg of tissue. Triplicates of 100 µL of homogenate were hydrolyzed in 100 µL of HCl (12N) for 3 h at 120 °C. Then, 10 µL of each sample  in duplicate were dried at 60 °C overnight in a 96-well plate. One of each duplicate was spiked with 4 µg of standard to correct for interference from endogenous compounds. Chloramine-T solution was added to each dried sample and incubated for 5 min followed by Ehrlich’s reagent, or DMAB , incubated at 60 °C for 90 min. The concentration of hydroxyproline was then determined by spectrophotometry at 560 nm and normalized to the mass of the sample. A standard curve was generated from known concentrations of hydroxyproline corrected from a background reading. The spiked sample readings were corrected with their corresponding non-spiked sample reading to calculate hydroxyproline µg/mg of muscle for each turkey. Collagen concentration was then calculated, assuming collagen weighs 7.5 times the measured hydroxyproline concentration [A hydroxyproline colorimetric assay kit  was used to determine the collagen content of the lateral gastrocnemius muscle from juvenile and adult domestic turkeys as well as adult wild turkeys. Hydroxyproline is an amino acid that is almost solely found in collagen and elastin in animals and can therefore be used as a direct measurement for collagen concentration within a tissue . Elastinntration .Fresh muscle samples taken from the mid-section of each LG, with aponeurosis intact for fiber orientation reference, were fixed in 10% formalin immediately after their removal, following previously published methods . SamplesThe samples were then prepared for imaging by first washing in 1% tannic acid for 3 h, followed by another three-hour wash in distilled water. They were then soaked in 1% Osmium tetroxide overnight. After two 30 min distilled water washes, the samples were dehydrated in an ethanol series: 30%, 50%, 70%, 95%, 100%, 100% and 100% for 15 min each. The ethanol was then eliminated via critical point drying  and the samples were mounted on an aluminum stub and coated in gold with a sputter coater . A scanning electron microscope  equipped with a lanthanum hexaboride gun was then used to view the tissue and images were collected with a Quartz PCI digital imaging system .Analysis of the SEM images was performed using ImageJ . The scaAverage fiber areas were found for other species to compare across a wide range of body mass . The average fiber cross-sectional area for the LG in the finch, pigeon, chucker, mallard, pheasant, chicken and goose were obtained from Snyder , coverint-test for age (juvenile domestic vs. adult domestic) and strain (adult domestic vs. adult wild) in JMP Pro 15.0 .The relationship between PCSA and body mass between strains, as well as the relationship between force and PCSA, were compared using standard major axis regressions in SMATR using R . The hyd−2, [−2 (r2 = 0.92) (−2 (p = 0.149). The LG PCSA increased with body mass in both strains, with wild turkey PCSA scaling with body mass0.93 (r2 = 0.35) and domestic turkey PCSA scaling with body mass0.79 (r2 = 0.97). The slopes were not significantly different between strains (p = 0.413). Scaling in the domestic turkey was significantly greater than the expected slope of 0.67 for geometric scaling  (p = 0.001). Measured peak muscle force in the domestic turkey LG was compared with predicted peak force, calculated from measured PCSA and a typical value of force/PCSA for vertebrate muscle . The pe = 0.92) A. This w= 0.003) B. For a p = 0.387, p = 0.0275, Collagen content, as measured by hydroxyproline assay, was compared between age groups and between strains . JuvenilEndomysium and perimysium collagen structure were characterized with SEM images. Qualitatively, there were visible differences in the collagen distribution and amount among the three turkey groups . The juv2, n = 2 birds totaling 848 fiber areas measured), which was significantly greater than adult domestic turkeys  (p = 0.013). Average adult domestic turkeys’ fiber areas were over 11 times larger than juvenile domestic turkeys’  (p < 0.0001). Based on fiber density estimates reported during the fiber area measurements of 1160 fibers mm−2 in the juvenile turkeys and 200 fibers mm−2 in the adult domestic turkeys, and using the PCSA of the whole muscle , and the regression was significant (p = 0.0002).The relationship between average LG  fiber cross-sectional areas and body mass was examined across a wide size range of species, providing context for averages observed in adult wild and domestic turkeys . Across Based on the selective regime that domestic turkeys have undergone, and known locomotor issues, we expected to find significant changes to the LG muscle’s structure and function. We found the differences in the LG muscle to be minimal between strains. The domestic turkey LG could produce the expected amount of force per area and the muscle fiber cross-sectional areas were not larger than the wild turkeys, as one might predict based on selection for increased meat production. We did find differences in the connective tissue of the LG extracellular matrix between wild and domestic turkeys, and these may have some functional consequences. However, even where we found similarities in the LG structure and function in these large turkeys, there are implications for locomotion and movement, which we address below. −2 The LG is most active during the stance phase of locomotion [The domestic turkey reaches a total body mass 3× that of a wild turkey, predominantly a result of increased muscle masses due to artificial selection . Howevercomotion ,47, and However, simple scaling issues still affect the domestic turkey. Despite the maintained peak muscle force performance, domestic turkey LG muscles are still relatively weaker than wild turkeys due to the large body masses domestic turkeys attain . The PCSConsumer preference for poultry meat is driven by appearance and texture . Meat teThe collagen concentration is not significantly different between juvenile domestic and adult domestic turkeys, indicating that the collagen content in a given volume of muscle is similar . This isIntramuscular fat deposits can also affect the structure and mechanical properties of the muscle. We saw evidence of intramuscular fat throughout the adult domestic turkey muscle samples C, and a Muscles can dramatically increase in size during growth and with activity, with whole muscle size being altered by an increase in the number of fibers or an increase in individual fiber size. Existing studies show increases in fiber size occur with activity and growth ,60, but Due to selection for larger muscle mass, we expected the domestic turkey fiber areas to be larger than the wild birds and for them to have more fibers within the muscle overall. Surprisingly, wild turkey muscle fibers turned out to have a greater average cross-sectional area than domestic turkey muscle fibers in this study . Since t0.80 [Domestic turkey LG muscles can be more than twice the size of wild turkey LG muscles and yet the domestic turkeys have slightly smaller fiber cross-sectional areas. One potential explanation for this surprising pattern is that the turkeys are running into some sort of absolute limit of muscle fiber area. To examine this idea, we gathered data on fiber size for a range of animals from the literature . The dat0.80 , and our0.80 ,67,68,69Selection for increased body mass and muscle mass has resulted in structural changes within the lateral gastrocnemius muscle of the domestic turkey. An increase in the number of muscle fibers is responsible for the increase in the LG cross-sectional area with body mass between the wild and domestic turkey strains. Collagen concentration was significantly decreased in the domestic turkey, which may reflect selection for tenderness. Despite these changes, the muscle was still able to produce the expected amount of force per physiological cross-sectional area. However, because the domestic turkeys reach much higher body masses, scaling has still resulted in domestic turkeys producing less force per unit body mass than wild turkeys. Force production is just one measure of muscle performance; the structural changes we observed in the intramuscular connective tissue of the domestic turkey muscle could have other functional implications for these large birds.