Recently, the incidence rate of renal fibrosis has been increasing worldwide, greatly increasing the burden on society. However, the diagnostic and therapeutic tools available for the disease are insufficient, necessitating the screening of potential biomarkers to predict renal fibrosis.Using the Gene Expression Omnibus (GEO) database, we obtained two gene array datasets (GSE76882 and GSE22459) from patients with renal fibrosis and healthy individuals. We identified differentially expressed genes (DEGs) between renal fibrosis and normal tissues and analyzed possible diagnostic biomarkers using machine learning. The diagnostic effect of the candidate markers was evaluated using receiver operating characteristic (ROC) curves and verified their expression using Reverse transcription quantitative polymerase chain reaction (RT-qPCR). The CIBERSORT algorithm was used to determine the proportions of 22 types of immune cells in patients with renal fibrosis, and the correlation between biomarker expression and the proportion of immune cells was studied. Finally, we developed an artificial neural network model of renal fibrosis.Four candidate genes namely DOCK2, SLC1A3, SOX9 and TARP were identified as biomarkers of renal fibrosis, with the area under the ROC curve (AUC) values higher than 0.75. Next, we verified the expression of these genes by RT-qPCR. Subsequently, we revealed the potential disorder of immune cells in the renal fibrosis group through CIBERSORT analysis and found that immune cells were highly correlated with the expression of candidate markers.DOCK2, SLC1A3, SOX9, and TARP were identified as potential diagnostic genes for renal fibrosis, and the most relevant immune cells were identified. Our findings provide potential biomarkers for the diagnosis of renal fibrosis. Renal fibrosis refers to the proliferation of fibrous tissue in the kidney due to trauma, infection, inflammation, and other factors, resulting in a gradual deterioration of renal function . Its patin vitro, indicating that ISG20 may play an important role in renal fibrosis  between the control samples and renal fibrosis samples were screened using the R package “limma” The screening conditions were: log fold change (FC) was greater than 2 and false discovery rate (fdr) was less than 0.05. Subsequently, we obtained the module with the highest correlation with renal fibrosis through weighted correlation network analysis (WGCNA)  and obtaLeast absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine-recursive feature elimination (SVM-RFE), were used to identify predictive genes for renal fibrosis –15. The Immune cells in renal fibrosis and control samples were evaluated using the CIBERSORT algorithm. Spearman rank correlation analysis was performed using the R package “ggplot2” to visualize the correlation between candidate biomarkers and various immune cells .GSEA was performed to analyze the potential biological functions of the candidate genes. A gene set named “c2.cp.kegg.v7.0. symbols.gmt” was downloaded from the Molecular Signatures Database (MSigDB) and selected as the reference gene set .To identify additional drugs targeting the candidate biomarkers for the treatment of renal fibrosis, we conducted a drug sensitivity analysis. The CellMiner database was used to download gene expression data and drug sensitivity data, and the R package “impute”, “limma”, “ggplot2” and “ggpubr” were used for drug sensitivity analysis .2. In the experimental group, we incubated the cells for 48h with recombinant TGF-β1 .HK-2 cells, a human renal tubular epithelial cell line, were obtained from The American Type Culture Collection  and maintained in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37°C with 5% COTotal RNA was extracted using the TRIzol reagent. Total RNA was reverse transcribed into the cDNA template according to the manufacturer’s protocol, and SYBR Green Real-Time PCR Master Mix Plus (Toyobo) was used to amplify the cDNA template. β-Actin was selected as an endogenous reference gene to normalize the mRNA levels. Primer sequences used for RT-qPCR are listed in First, the DEG expression data were converted to a Gene Score table based on expression levels. The medians of all the sample expression values and the expression values of a single gene in a given sample were compared. If the expression value of the upregulated gene was greater than 0, it was given a value of 1; otherwise, it was given a value of 0. Likewise, if the expression value of the downregulated gene was higher, it was given a value of 0; otherwise, it was given a value of 1. Renal fibrosis was the outcome variable; cases were assigned a value of 1, whereas controls were assigned a value of 0. An artificial neural network model was visualized using the R package neuralnet  based onThe flow chart of this study was shown in https://cn.string-db.org/). A protein-protein interaction (PPI) network  curves for the four candidate biomarkers and found that DOCK2, SLC1A3, SOX9, and TARP had good diagnostic efficacy, with the area under the ROC curve (AUC) values of 0.886, 0.815, 0.843, and 0.797, respectively , activated NK cells, macrophages M2 and resting mast cells was decreased  were used to screen for potential diagnostic biomarkers for renal fibrosis. Four genes with overlapping features  were finally selected. The roles of these genes in fibrotic diseases have been reported previously. For instance, Xia et al. found that DOCK2 deficiency could weaken bleomycin-induced pulmonary fibrosis through TGF-β signaling pathway . Recent The chemokine signaling pathway is implicated in renal fibrosis. Renal fibroblasts secrete CCL2, CCL3 and other inflammatory chemokines, which drive various inflammatory cells to the lesion site, thereby promoting the formation of renal interstitial fibrosis , 27. A lRenal fibrosis is often accompanied by a series of changes in the proportion and function of immune cells. According to Purvi et al., IL-17 secretion may participate in the pathogenesis of renal fibrosis after AKI via neutrophil recruitment . In obstGiven that renal fibrosis and cancer share a common feature of enhanced epithelial mesenchymal transition (EMT), as well as the inhibitory effects of many anti-tumor drugs on EMT, we analyzed the relationship between these drug sensitivity and model genes to provide possible direction for the treatment of renal fibrosis. The results showed that DOCK2 and TARP were positively correlated with most drugs. These findings lay the foundation for anti-fibrosis drugs.Although this study constructed a novel neural network model to predict renal fibrosis based on high-throughput sequencing data, it had several limitations. Firstly, this was a retrospective study with a relatively small sample size, which remains unreliable. Therefore, a prospective study with a larger sample size is warranted.In summary, we determined four potential diagnostic biomarkers in the peripheral blood of patients with renal fibrosis through machine learning and comprehensively analyzed their diagnostic value, drug sensitivity, and correlation with immune cells. These findings will help improve the diagnosis and treatment of renal fibrosis.The original contributions presented in the study are included in the article/MJ and YG designed and directed the study. KH wrote the manuscript. KC and YM performed data collection and curation. KH performed RT-qPCR experiments. All authors have read and agreed to the published the version of the manuscript. All authors contributed to the article and approved the submitted version.


Fruits and vegetables are important sources of nutrients such as vitamins, minerals, and bioactive compounds, which provide many health benefits. However, due to suboptimal postharvest management, large quantities of fresh or fresh-cut fruit and vegetables lose their quality and nutritional value before they reach the consumer.Advances in Post-harvest Preservation Technology, belonging to the Section “Food Packaging and Preservation”, 14 research and 2 review articles are included. Five research topics are addressed: (i) storage and treatment; (ii) packaging and edible coating; (iii) techniques or markers for determining fruit quality; (iv) prediction models; and (v) postharvest technologies and preservation strategies.In this Special Issue, Lycium barbarum L.) stored at different temperatures  for up to 12 d, highlighting significant differences concerning the fruit’s phytochemical attributes , and anthocyanin content). This study revealed that goji fruit is optimally stored at a temperature of 5 °C for 9 d. At this temperature, physiological disorders (such as pitting and shriveling) were reduced, while the overall sensorial and nutritional quality attributes, including vitamin C levels, total soluble solid content, and antioxidant activity, were preserved. However, additional technologies such as modified atmosphere packaging may be required to better control decay and extend shelf life. Ma et al. [Juglans regia L. cv. Xiangling, with a green husk, shell, and fresh kernels) stored between 0 and −20 °C for 10 months. The results showed that the walnuts with a green husk presented higher-quality kernels compared to the other forms of walnuts analyzed when stored at 0 °C for 3 months. Whereas at a freezing temperature (−20 °C) and during long storage, the walnuts with shells showed advantages with respect to maintaining fatty acid content, total phenols, and total ascorbic acid concentrations compared with other forms of walnuts. To preserve the quality of fresh-cut cauliflower (Brassica oleracea L. botrytis cv. Xuebai), vegetables were immersed in a solution containing calcium chloride (CaCl2) at a 2% concentration and at different temperatures  for 10 min [2 and high temperature (40 °C) maintained a higher firmness value, preventing the browning and yellowing of fresh-cut cauliflower florets. This treatment was optimal for preventing reductions in ascorbic acid content and total glucosinolates and inhibiting the activity of polygalacturonase and the softening of cauliflower florets compared to the other treatments, particularly with respect to the control sample.Pertaining to the first topic, the article published by Fatchurrahman et al.  reports N-acetyl-5-methoxytryptamine) applied to preserve and extend the shelf life of mangos and zucchinis. In the first study, a trial measuring the response of on two mango (Mangifera indica L.) cultivars to exogenous melatonin (1000 mol L−1) was conducted by Njie et al. [Two papers reported the results of treatments with melatonin . Zucchinis were stored at 4 °C for 15 d and an additional 2 d at 20 °C. For the zucchinis treated with melatonin + 1 MCP, weight loss and signs of chilling damage were reduced compared to the other treatments and the control, maintaining a higher degree of fruit firmness throughout cold storage. Moreover, for each sampling day, the combined treatment (1-MCP + melatonin) effected the lowest values of malondialdehyde and electrolyte leakage. The combined treatment only reduced the respiration rate for 9 d, while ethylene production was reduced only by the treatment with 1-MCP alone.Medina-Santamarina et al.  reportedWhile stored at 4 °C, the total soluble solids and total chlorophyll content in the combined treatment were comparable with 1-MCP application. The authors highlighted that the proposed combined treatment could be a promising tool for increasing the storability of zucchinis at suboptimal temperatures.Prunus persica L. cv. Xiahui 8), treatment with 1-MCP, alone or in combination with nitric oxide, was proposed [In another study, to retard peach senescence (proposed . At the Allium cepa L.), corn oil, salt, sumac (Rhus coriaria L.), and lactic acid) on the quality traits of fresh Za’atar (Origanum syriacum) sealed in vacuum bags and stored at 2–4 °C for 42 d. Microbial counts, color, and other sensory attributes were analyzed every 7 d. The study showed that the addition of lactic acid induced a strong preservative effect against aerobic and anaerobic bacteria. Moreover, the addition of sumac improved the preservation of vacuum-packaged oregano stored in refrigerated conditions.The research conducted by Mudalal et al.  revealedRubus idaeus L. cv. ‘Kweli’). To extend the shelf life of this perishable fruit, pads produced with chitosan, green tea, or rosemary ethanolic extracts were incorporated as natural antifungal agents. Sealed fruit trays were packaged under an air atmosphere using polyacid lactic film and stored at 4 °C for 14 d. At the end of the storage period, reductions in the growth and decay of spoiled fruit of 5% and 13% were observed for chitonsan + rosemery and chitonsan + green tea, respectively. Conversely, for the fruit packets without pads, these percentages were equal to 40 and 80% after 7 and 14 d, respectively. The use of polyacid lactic film reduces weight loss, thus preserving the firmness of raspberries, particularly when using pads with green tea or rosemary ethanolic extracts. Total phenols and ascorbic acid content were better preserved in the packages with film pads containing one of each extract. The results highlighted how the application of chitosan pads + rosemary ethanolic extracts could be applied to other soft fruit to increase their marketability.Regarding the second topic, (ii) packaging and edible coating, Vieira et al.  reportedPhoenix dactylifera L. cv Barhi) fruit in combination with extracts of olive cake and orange peel in two different concentrations (1 or 2%) during storage at 4 °C for 28 d [As an edible coating, chitosan was also used to preserve the quality attributes of fresh date (for 28 d . When thDaucus carota cv. Nantes) stored at 4 °C for 14 d. The application of lemon extract preserved the fresh-cut carrots’ physiological parameters, thereby delaying their senescence. The carrots showed limited changes in color and white-blush and a good degree of firmness throughout storage due to the presence of calcium chloride in the coating’s formulation. The lemon extract by-product improved the microbiological stability of the minimally processed carrots, showing the lowest value of total bacterial activity after 7 d, while a mild increase was observed following the end of the storage period. Total carotenoids, phenolic content, and antioxidant activity values exhibited a similar trend during storage for all treatments, while higher levels were measured in the fresh-cut carrots treated with lemon extract.Imeneo et al.  studied Fragaria x ananassa Duch. cv Sabrosa). The first one, proposed by Cozzolino et al. [Regarding topic (iii) concerning the techniques or markers for determining fruit quality, two papers focused their activity on Candonga strawberries  during the ripening season. Multiplex® 3  equipped with a portable optical sensor was used for analysis. The study focused on the measurement of skin anthocyanin using two fluorescence indexes, namely, ANTH_RG (chlorophyll fluorescence excited with red and green lights) and FERARI (fluorescence excitation ratio anthocyanin relative index), and other indices concerning the total flavonoid, nitrogen, and chlorophyll content. All measurements were correlated with the principal quality parameters  obtained by applying common analysis techniques. The results highlighted an important relationship between the total anthocyanins (measured via spectrophotometry) and ANTH_RG and FERARI indices with R2 values equal to 0.96 and 0.87, respectively. The main result obtained by the researchers was a regression equation developed using the ANTH_RG index (measured by Multiplex® 3) and skin anthocyanin content , which allows one to estimate the previously mentioned parameters directly in-vineyard in rapid mode and without damaging the plant material.Hamie et al.  studied Citrus sinensis L. cv Rustenburg) in order to develop shelf-life prediction models with which to enable the use of a First Expired–First Out (FEFO) logistics strategy [2 of 0.891. Thus, in the future, the development of accurate shelf-life prediction models should contribute to optimizing the FEFO logistic management system and thus promote more efficient inventory management and loss reduction. Postharvest storage and preservation strategies for extending the storage life of the king oyster mushroom (Pleurotus eryngii D.C.) were described in the review article written by Gou et al. [Pleurotus eryngii were the use of the lactic acid fermentation process and nanoparticles with which to encapsulate bioactive substances. According to Gou et al. [Pleurotus eryngii should focus on combining new and traditional technologies to improve postharvest quality. Among such techniques, it was stressed that radiation treatment with 1-MCP alongside a nano-packaging treatment, safe and efficient sterilization, microwaving, and the use of low-pressure electrostatic fields and low-temperature plasma sterilization equipment could be successfully applied during the storage and distribution of king oyster mushrooms. In the study conducted by Palumbo et al. [Owoyemi et al. conducted phenotyping analysis of the effects of various preharvest and postharvest quality levels of navel oranges (strategy . During u et al. . Many stu et al. , future o et al. , the latIn conclusion, the papers proposed in this Special Issue, which were written within different European and national projects or promoted by private enterprises, report innovative results that extend our knowledge of the postharvest preservation technology applied to fruit and vegetables that are either fresh or minimally processed. The Guest Editors thank all authors that have contributed to enriching the Special Issue and hope that the results presented might constitute a stimulus for further studies.


We hypothesized that diffuse TBI would alter circulating GH levels because of damage to the hypothalamus, specifically somatostatin neurons. Data were analyzed with generalized linear and mixed effects models with fixed effects interactions between the injury and time. Despite similar growth rates over time with age, TBI rats weighed less than shams at 18 DPI , which is around the onset of puberty. Compared to shams, GH levels were lower in the TBI group during the acute period  but higher in the TBI group during the chronic period . Although not statistically significant, TBI-induced differences in GH had large standardized effect sizes, indicating biological significance. The mean number of hypothalamic somatostatin neurons (an inhibitor of GH) positively predicted GH levels in the hypothalamus but did not predict GH levels in the somatosensory cortex. Understanding TBI-induced alterations in the GH axis may identify therapeutic targets to improve the quality of life of pediatric survivors of TBI.Traumatic brain injury (TBI) can damage the hypothalamus and cause improper activation of the growth hormone (GH) axis, leading to growth hormone deficiency (GHD). GHD is one of the most prevalent endocrinopathies following TBI in adults; however, the extent to which GHD affects juveniles remains understudied. We used postnatal day 17 rats ( Annually, approximately 70 million people worldwide suffer from a traumatic brain injury (TBI) . IndividDespite the primary injury from a TBI being irreversible, subsequent injury processes occur in a delayed manner and may be responsive to medical management. The mechanical forces of TBI and the resultant secondary injury mechanisms lead to neuropathology and cellular damage to internal structures of the brain, including the hypothalamus and pituitary  axis results in linear body growth and changes in body size and composition , the preClinical studies indicate that over a 1-year span patients who suffer a TBI are at a higher risk for developing an endocrine system disorder than those who have not sustained a TBI. We have previously shown that specific to the pediatric population, children with a TBI diagnosis have 3.22× the risk of a subsequent endocrine disorder diagnosis compared with the general pediatric population . Predetermined exclusion criteria were excluding any rat that lost >20% of their body weight or had unmanageable pain. No rats in this study met those criteria, so none were excluded. Predetermined inclusion criteria included a righting reflex time >120 s and no breach of the dura. All plasma samples and slides were relabeled with a code by an investigator not associated with the study to ensure that investigators were blinded to the experimental conditions.All animal studies were conducted in accordance with the guidelines established by the Institutional Animal Care and Use Committee (IACUC) at the University of Arizona and the NIH guidelines for the care and use of laboratory animals. Studies are reported following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines  were used for all experiments. Rats were housed in a 12 h light:12 h darkness cycle at a constant temperature (23°C ± 2°C) with food and water available eriments . WeightsFor surgery, all rats were administered 5% isoflurane in 100% oxygen for 5 min and then secured in a stereotaxic frame. Anesthetization was maintained with continuous isoflurane delivery at 2.5% via nosecone. A midline incision was made and a craniectomy (outer diameter 3mm) was trephined midway between bregma and lambda  with methods we have previously described for postnatal day 17 rats . After rBlood was collected via cardiac puncture at predetermined time points post injury  solution (Vectastain ABC kit PK-6100) for 30 min. Slides were washed in PBS + 0.1% Tween 20 and then incubated in DAB solution (from Vector DAB peroxidase substrate kit SK-4100) for 10 min and, following this, slides were immediately placed in water. The brain tissue was dehydrated in ethanol , cleared with citrosolve, and coverslips were added using dibutylphthalate polystyrene xylene mounting medium.Antigen retrieval was performed using sodium citrate buffer (pH 6.0). Slides were then washed in 1× PBS. Hydrophobic barrier pen was used around the perimeter of the slide, and slides were placed in a humidity chamber. Slides were incubated in blocking solution  for 60 min. Following blocking, slides were incubated in primary antibody solution  overnight at 4ºC. Slides were then washed in 1× PBS + 0.1% Tween 20. Slides were incubated in secondary antibody solution (biotinylated horse anti-rabbit IgG (H + L); vector BA-1100; at 1:250 concentration in 4% NDS and 0.4% Triton 100 in 1× PBS) for 60 min. Slides were washed in 1× PBS + 0.1% Tween 20. Endogenous peroxidases were blocked in 200 mL 1× PBS + 8 mL HImages of stained tissue were taken using Zeiss Imager A2 microscope via AxioCam MRc5 digital camera and Neurolucida 360 software. For somatostatin neurons, quantification was completed from images ranging from −1.65 to −3.60 in reference to Bregma using a modified version of unbiased stereology. The regions of interest (ROI) were the medial preoptic nucleus, from rostral to the decussation of the anterior commissure, through the caudal end of the decussation of the anterior commissure (Plasma GH levels were quantified using enzyme-linked immunosorbent assay (ELISA) kits . GH samples were run in triplicates following the manufacturer’s instructions.We used generalized linear models to investigate differences in righting reflex times, apnea times, body weights, growth hormone levels, and number of neurons between sham and injured rats across time (DPI). Righting reflex times and number of neurons were overdispersed counts, so we specified negative-binomial error distributions in all models that contained those outcome measures . Apnea tλ) of body weight that naturally occurs with increasing pubescent rat age differed between sham and injury. To test this and quantify the rate of increase, we fit a generalized linear mixed effects model with random intercepts for individual rats . We based inferences on a combination of model coefficient estimates (β), P-values from posthoc multiple comparisons tests corrected via Tukey’s method, predicted marginal (fixed effects models) and conditional (mixed effects models) effects and their 95% confidence intervals (d).We fit both generalized linear and generalized linear mixed models using the package glmmTMB in the R statistical computing environment .There were no statistically significant differences in the surgery weights of rats assigned to the sham group compared to rats assigned to the TBI group , 18 DPI ; 25 DPI , and 43 DPI . Compared to postnatal 18 days, GH levels were comparable at postnatal 24 days , postnatal 42 days , and postnatal 60 days . GH levels at postnatal 60 days were also significantly elevated compared to postnatal 24 days  or 43 DPI . Similarly, differences in somatostatin neurons in the somatosensory cortex between sham and TBI rats were not supported at 1 DPI , 7 DPI , or 43 DPI  but was significantly higher at postnatal 60 days , and the mean number of somatostatin neurons at postnatal 60 days was higher than at postnatal 24 days . In contrast, the mean number of somatostatin neurons in the somatosensory cortex decreased over time with animal age  and postnatal 60 days , and the mean number of neurons at postnatal 24 days was lower than at postnatal 60 days .The mean number of somatostatin neurons in both the medial preoptic area and somatosensory cortex was calculated for each rat . Althougimal age ; compareβ = 0.65; P = 0.55; β = −0.03; P = 0.33), and the number of somatostatin neurons in the somatosensory cortex was not a predictor of growth hormone levels . Considerable variation exists in the onset of puberty in male rats, but postnatal day 35 is typically when the onset of puberty occurs . AdditioFollowing both TBI and sham conditions, GH levels increased over time with animal age. Although longitudinal measurements of post-TBI GH levels have yet to be recorded in rats, many clinical studies provide ancillary support for the general trend that we observed. Specifically, GH production tends to vastly increase during puberty, and then gradually declines during adulthood . In the Much of the current literature indicates that GHD represents the majority of hormonal changes observed in the acute postinjury period . The disAt 1 DPI, the number of somatostatin neurons in the medial preoptic area of the hypothalamus acutely decreased in rats subjected to diffuse TBI compared to uninjured shams. This decrease in number of somatostatin neurons at 1 DPI directly coincides with the acute decrease in GH that we observed in brain injured rats. Although the number of somatostatin neurons in the somatosensory cortex also decreased at 1 DPI, this change was not significant. It is plausible that the acute decrease in neurons observed in the current study is not specific to somatostatin neurons but is a result of a general loss of neurons or neuronal complexity in the acute phase post TBI .Interestingly, we found changes in somatostatin neurons over time with animal age were brain region-specific. Somatostatin neuron number increased over time with animal age in the medial preoptic nucleus of the hypothalamus. In contrast, the number of somatostatin neurons in the somatosensory cortex decreased over time with animal age. We have previously observed significant neuropathology, identified by hyperintense deposition of argyrophilic reaction product , in the somatosensory cortex of rats subjected to midline fluid percussion injury  and insulin-like growth factor 1 (IGF-1), were not measured. Both GHRH and IGF-1 have major contributions to the regulation of GH and therefore are likely to contribute to GHD. Historically, both IGF-1 and GHRH measurements have been employed as diagnostic criteria of GHD , the Valley Research Partnership VRP43 223010 , and the Brain Injury Association of Americahttp://dx.doi.org/10.13039/100014906 Seed Grant . ST was supported by the National Institutes of Healthhttp://dx.doi.org/10.13039/100000002 Workforce Inclusion in Neuroscience through Undergraduate Research Experience (WINURE) at Arizona State Universityhttp://dx.doi.org/10.13039/100007482. NC and GSM were supported by the University of Coloradohttp://dx.doi.org/10.13039/100010174 Boulder Undergraduate Research Opportunities Program (UROP) fellowship.These experimental studies were funded by the JO assisted with conceptualization, data collection, and led writing of the manuscript. ST, GR, GSM, NC, MM, and TG assisted with experimental design, data collection, and editing the manuscript. SMM led statistical analyses and assisted with reviewing and editing the manuscript. RR was responsible for conceptualization, experimental design, funding acquisition, formal analysis, writing, and reviewing and editing the manuscript.


Panicum virgatum L.) is a promising perennial bioenergy crop that achieves high yields with relatively low nutrient and energy inputs. Modification of cell wall composition for reduced recalcitrance can lower the costs of deconstructing biomass to fermentable sugars and other intermediates. We have engineered overexpression of OsAT10, encoding a rice BAHD acyltransferase and QsuB, encoding dehydroshikimate dehydratase from Corynebacterium glutamicum, to enhance saccharification efficiency in switchgrass. These engineering strategies demonstrated low lignin content, low ferulic acid esters, and increased saccharification yield during greenhouse studies in switchgrass and other plant species. In this work, transgenic switchgrass plants overexpressing either OsAT10 or QsuB were tested in the field in Davis, California, USA for three growing seasons. No significant differences in the content of lignin and cell wall-bound p-coumaric acid or ferulic acid were detected in transgenic OsAT10 lines compared with the untransformed Alamo control variety. However, the transgenic overexpressing QsuB lines had increased biomass yield and slightly increased biomass saccharification properties compared to the control plants. This work demonstrates good performance of engineered plants in the field, and also shows that the cell wall changes in the greenhouse were not replicated in the field, emphasizing the need to validate engineered plants under relevant field conditions.Switchgrass ( Such biorefinery processes can reduce the need for petroleum-derived fuels and chemicals. To enable this aspect of a bioeconomy, it is essential to have high yielding crops for biomass production that can be grown with low inputs and are optimized for downstream processing . LignoceMuch research has focused on the modification by breeding or engineering of bioenergy crops to optimize their suitability for downstream processing, e.g. by reducing lignin content, since lignin is the main biomass component that limits deconstruction of biomass into convertible sugars . There aQsuB gene from C. glutamicum, which encodes a dehydroshikimate dehydratase and leads to a reduction in precursors of lignin biosynthesis. This approach leads to strong reduction in lignin in Arabidopsis and poplar, and moderate lignin reduction in switchgrass  expressing OsAT10 and QsuB under control of the maize Ubiquitin1 (pZmUbi-1) and sugarcane O-methyltransferase (pShOMT) promoter, respectively, were generated by Agrobacterium-mediated transformation as previously described . The plots were fully irrigated to match expected crop evapotranspiration at this site so that no water stress occurred. Urea was applied in April and after each harvest at a total level each year of 404, 252, and 269 kg N/ha. The experimental plot design was a randomized complete block design with four replicates for each transgenic line and controls . Each replicate was planted to 3 rows per plot and 10 plants per row of the experimental plants, which were surrounded by non-transgenic border plants . Plants were vegetatively propagated in a greenhouse and were transplanted under permit to a field site at UC Davis. The soil was a Yolo Silt Loam soil series classified as fine-silty, mixed, superactive, nonacid, thermic Mollic Xerofluents  and milled into a fine powder using a Mixer Mill MM 400  and stainless-steel balls. Ball-milled biomass (1 g) was sequentially extracted by sonication (20 min) with water (twice), 80% (v:v) ethanol (five times), acetone (once), chloroform-methanol  and acetone (once). Klason lignin was determined using the standard NREL biomass procedure . Monosacescribed  and quanescribed .2.4−1). The chromatograph was operated at a split ratio of 10 and the program was set at 50°C for 1 min followed by ramping to 300°C at 20°C min−1 and finally maintained at 300°C for 10 min. Released products of S and G origin were identified on the basis of their mass spectra using the NIST08 mass spectrum library and quantified from the chromatogram using the peak area.Pyrolysis coupled with gas chromatography mass spectrometry was used to determine lignin S/G ratio on cell wall residues, as previously described . Specifi2.5For saccharification assays, 10 mg of extracted biomass was pretreated with hot water  followed by a 72-h enzymatic hydrolysis at 50 ˚C (1200 rpm) using 1% w/w Cellic CTec2 enzyme mixture  in a total volume of 1 ml as previously described . A 10-µl2.62 and temperature were held at 400 ppm and 36 ˚C, respectively, with light provided using a LiCor 6400-40 LCF leaf chamber. Measurements were done on two plants in each replicate plot for Alamo control, FT8 and QsuB13 . The light intensity was set initially at 2000 µmol m-2 s-1 and then stepped down to 1000, 300 and 0 µmol m-2 s-1. At each point, net CO2 assimilation and transpiration was logged after a stable reading was observed.Photosynthesis rate was measured in the field on July 11, 2018, using a Li-6400/XT Portable Photosynthesis System . Chamber CO2.7https://www.foragetesting.org/reference-methods) for Neutral Detergent Fiber (NDF) analysis. In vivo (in sacco) measurements utilizing nylon bags inserted into live cows measured disappearance of the NDF fraction in rumen fluid at selected time points. Wet chemistry was conducted at Cumberland analytical Lab . Near-infrared reflectance spectra were collected using a scanning monochromator . Robust calibrations for prediction of crude protein (CP), NDF, Acid Detergent Fiber (ADF), lignin (Acid Detergent Lignin), digestibility estimates, and forage quality parameters were generated using the 2018 Grass Hay calibration developed by the NIRS Forage and Feed Testing Consortium . Data were statistically analyzed using SAS to test for the influence of entry (variety), cut, and the interaction between entry and cut.Switchgrass samples were ground to 1 mm cyclone screen and forage quality parameters measured using wet chemistry and Near Infrared Spectroscopy (NIRS) methods from the 2018 full season sample set. First cut samples were measured utilizing wet chemistry methods  was observed  that is required for the 3-hydroxylation of 4-coumaroyl-CoA to caffeoyl-CoA. In dicots, HCT is an essential enzyme for lignin biosynthesis and HCT mutants have highly reduced growth. Plants have another enzyme that can 3-hydroxylate p-coumaric acid to caffeate  and ferulic acid (about 20%) that had been observed in the greenhouse for the QsuB  and FT p sorghum , Arabido sorghum , and pop sorghum . In Arabcaffeate , and whi content .In this field study, the improvements of biomass saccharification in transgenics were much smaller than those previously observed in plants grown in the greenhouse. However, the saccharification results correlate with the lignin and ferulic acid contents, and the very small effect in the field-grown QsuB plants is consistent with the lack of significant difference in biomass composition. The results underscore that it is essential to evaluate plant varieties, including transgenic plants, under realistic growth conditions in the field. Plants must not only be grown in the field but also managed according to standard agronomic practice for the crop in terms of e.g. planting density, fertilizers and irrigation. Pest and disease can obviously be an additional factor in field experiments, but we did not observe any signs of pest or disease in the plants. Switchgrass grown in California generally have few issues with disease. While it is not unusual to find differences in phenotype comparisons between controlled conditions and field studies, it is not clear what are the most important environmental factors that drive the different outcomes in this particular study. Less pronounced reduction in lignin in lignin-engineered trees grown in the field compared to the greenhouse has been observed in several studies (recently reviewed in ). These QsuB leads to accumulation of protocatechuate in the plants at low levels (~0.3 mg protocatechuate per gram biomass was observed in transgenics from the greenhouse), and the potential role of this aromatic during plant development remains unknown. Also, the QsuB expression may lead to other changes in the intermediates in the shikimate pathway, and it is possible that such changes lead to alteration e.g., in the synthesis of plant hormones. It is also possible that the metabolic changes work indirectly by altering exudate profiles and the rhizosphere microbiome. These possibilities will be the subject of future studies.While the compositional changes that were originally targeted with the engineering strategy did not lead to significant changes when the plants were grown in the field, we found an unexpected biomass yield improvement in the QsuB lines. This is consistent with what we have observed for QsuB sorghum lines in the field . The QsuThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.ML coordinated the field trial permitting and the analytic work. HS, CS, and DP planned the field trials and supervised the study. CL, Y-CC, and JO analyzed biomass properties. CB and DP conducted the field trials and performed forage composition analysis. AE generated the QsuB plants and supervised biomass analysis. JM supervised biomass analysis and advised on the field trials. PR and GL generated the AT10 plants. HS performed photosynthesis measurements and wrote the manuscript draft with contributions from all authors. All authors read and approved the final manuscript. All authors contributed to the article.


P < 0.01) UFD, TD, FTL and RTL. In addition, udder shape affected (P < 0.01) UW, UFD, URD, ULV and DFT. None of the other studied factors affected milk production traits. The bowl udder shape (P < 0.01) yielded a high total milk yield (3267.19 kg), adjusted milk yield (2443.01 kg) and lactation length (480.70 d) compared to other udder shapes. The genetic correlations of UW with total milk yield and persistency were strongly positive . However, strong negative genetic correlations were found between UW with peak milk yield and lactation length , between RTL with peak milk yield (− 0.92) and DRT with persistency (− 0.79). As found from the stepwise multiple regression, UW and URD can be used as good indicators for predicting milk yield and lactation length. Additionally, this study spotlights the genetic background of udder characteristics based on reliable studies and the QTL database for cattle as a first step toward applying this knowledge side by side with phenotypic traits to improve the productivity of the Holstein breed under subtropical conditions.The aggregate udder shape , udder measurements  and teat measurements  were measured on 1300 Friesian cows located in a commercial farm under subtropical conditions (Egypt) to appraise udder and teats status and to evaluate the possible relationships with some milk production characteristics in conjunction with udder shape, age at first calving, sire and inbreeding effects on udder morphological traits and milk production ability. For such an available sample size, parity had affected ( The conformation of cow udder used to be one of the important criteria for predicting milk production performance4. Udder measurements are important for the prognosis of udder health status and functionality of milk production in dairy cows. The morphology and anatomy of cow udder has long been a subject for curious selection to improve the efficiency of milk production5. In addition, the teats of dairy animals are an important part of the udder, which is attached with a milking cluster and meantime, serves the roles of both a valve regulating the outflow of milk and of a natural barrier for exogenous infections7, teat shape and dimensions have no less emphasis in that regard5. Poor udder and teat conformations form a management challenge for dairy cow commercial producers. However, selection against poor teats and udders increases the profit potential by increasing performance, longevity and showing the ability of the cow11. Moreover, significant relationships were monitored between dimensions and formation of teats and teat canal length with milk-ability and milk flow speed traits13.Concerning the productivity evaluation of dairy cattle, udder conformation are important elements for the assessment of milk production14 reported that udder size and shape conformation traits could play a vital role in the suitability for easy milking and economical milk production and should be considered when selecting dairy cows. A large sized udder with a large proportion of glandular tissue and a symmetrical shape is an asset to a milk animal16. Seykora and McDanel17 found that udder depth and teat-end shape have been associated with udder health and reducing frequencies of cows with deep udders and flat disks or inverted teat ends may reduce mastitis incidence. In addition, streak canal diameter was negatively correlated with udder health, though it is difficult to measure18.Bhuiyan et al.21 and that the teat canal length possesses a significant effect on the flow of milk through the teat canal22. Klein et al.23 recommend long and narrow teats to improve udder health. The teat canal length had a significant effect on milk flow characteristics such as daily milk yield, average milk flow, maximum milk flow and somatic cell score, at different measurement times25. Additionally, the length and morphology of the teat canal may have an effect on the development of udder infection and defence mechanisms25. Factors that have been associated with the quality of these mechanisms are udder depth, fore udder attachment, teat length, teat shape, and milk-ability26. It is likely that high udders with good attachments and small teats are less prone to teat lesions27. Relationships between teat shape and measurements with milk yield should be regarded as criteria for the selection of dairy cattle28. Wilmink29 and Sabuncuoglu et al.30 proposed that in dairy cattle selection, conformation traits, udder depth, suspension of fore quarters and teat location should be considered in addition to milk performance. Correlations between traits describing udder form and milk performance indicate clearly that udder depths, distance to floor, hind udder attachment, udder band, teat placement and teat size are all of economic relevant and useful to describe udder functionality. These traits can be recorded with a high degree of accuracy and repeatability32. Type traits are recorded relatively early in life of animals and are medium to highly heritable33. Heritability of udder morphology is to high34, a single score during the lifetime of a cow may be adequate for selection and which makes selection relatively easier and more efficient. Udder height was a good predictor of lactation performance35. Moreover, differences in udder shape and size were reported to be heritable15. Daughters of highly proven milk bulls possessed greater distance between teats, greater perimeters and larger areas of the udder floor and udder length were significantly correlated with milk yield36. Most dairy cattle breeding objectives worldwide have focused exclusively on production traits paying no attention to the severe deterioration in production properties caused, mainly by the unfavourable phenotypic and genetic correlations between linear type and milk production traits37. On the other side, there are genomic regions, that involve quantitative traits locus (QTLs), candidate genes and significant single-nucleotide-polymorphisms (SNPs) related to the udder and teat traits in dairy cattle38.Many researchers agree that there is a linear relationship between teat distances and milk yieldThe present study aimed to evaluate udder and teat characteristics in order to predict their relationship with milk production traits. The effects of udder shape, sire, parity, inbreeding and age at first calving on udder and teats measurements were also studied, with a spotlight on the genetic background of concerning traits.This experimental protocol was approved by the Ethics Committee of Damanhur University under approval number DFU-2023-2.This study was conducted on 1300 pure Friesian cows located in a commercial farm , in Wadi-El-Natrun, Al-Beheira Governorate, Egypt. Cows were housed free in open semi-shaded yards, nourished under the prevailing feeding conditions according to the NRC requirements and milked twice daily.39, udder heights (UH); distance from the ground to udder floor in front of the front or behind rear teat, teat diameters (TD); diameters of front teat or rear teat measured at mid-point of each teat length from teat orifice to base of udder, front teat length (FTL); length of front teat from teat orifice to base of udder, rear teat length (RTL); length of rear teat from teat orifice to base of udder, distance between front teats (DFT); distances between front teats before milking and at mid teat length, distance between rear teats (DRT); distances between rear teats before milking and at mid teat length  were taken monthly on a fixed day throughout the lactation period one hour before the evening milking. The udder and teat measurements were udder width (UW); distance between right and left sides of the udder at the widest point, udder front depth (UFD); distance from the merging point of fore udder with the abdomen to a point in front of the fore teats at the level of the udder base, udder rear depth (URD); distance from the bottom of the vulva to the base of the rear udder, udder levelness (ULV); the difference between the rear and front udder heightsgth Fig. .Figure 140 as ratio of 305d-MY / (PMY * 305). Udders were classified into bowl, cup and round shapes.Recorded milk production traits were total milk yield , adjusted milk yield , peak milk yield , lactation length  and persistency  which was calculated according to Lean et al.https://www.fao.org), (2) Animal QTL-Database:  and (3) Genome-Informatics-Resources:  for further and complementary future studies.According to reliable investigations, many genome regions, candidate genes, SNPs and QTLs have been reported to be correlated with several udder characteristics in different species including cattle. In this regard, the genetic background for udder characteristics in the cattle has been investigated utilizing; (1) FAO-Database . To study the factors affecting udder or teat characteristics, MIXED procedure of SAS 9.0 (2009)41 was used according to the following model:Yijklmn = any of the udder or teat measurements, µ = the overall mean, Si = the random effect of ith sire (i = 1:180), Pj = the fixed effect of jth parity (j = 1:5), Ik = the fixed effect of kth inbreeding (inbred or non-inbred), Ul = the fixed effect of lth udder shape , bm = a regression coefficient of each udder and teat measurement on the independent continuous variable of age at first calving specific to the teat or udder measurement and eijklmn = the residual error.All sets of data were tested for normality with the Shapiro–Wilk test from the UNIVARIATE procedure SAS 9.0 (2009)41 according to the following equation42:σ2S = the variance of sire and σ2e = the variance of error. Simple regressions and correlations between udder or teat measurements and all milk production traits were calculated. Besides, the stepwise multiple regression analysis of milk production traits on udder and teat measurements were calculated using STEPWISE procedure of SAS41 to determine the best regression model.Significant differences among means within each factor were tested using Duncan test. The heritability values of each udder or teat measurement was estimated using variance components The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Also, this experimental protocol was approved by the Ethics Committee of Damanhur University under approval number DFU-2023-2.Means, standard deviations and coefficients of variation of udder and teat characteristics in different udder shape groups are shown in Table P > 0.01) on UW, UFD, URD, ULV or UH, but parity had an effect (P < 0.01) on UFD which was the highest (22.58 cm) in parity 3. Udder shape had effects (P < 0.01) on UW, UFD, URD and ULV with highest values of UW, UFD and URD  obtained for bowl shaped udders but the highest values of ULV was obtained in cases of round (− 3.48 cm) and cup shaped (− 3.03 cm).Least squares means and standard errors for UW, UFD, URD, ULV and UH as affected by sire, inbreeding status, parity, udder shape and age at first calving are presented in Table P > 0.01) on TD, FTL, RTL, DFT and DRT. Parity had effects (P < 0.01) on TD, FTL and RTL with the highest values obtained in parities 3, 4 and 5. Udder shape had no effects on teat measurements except DFT which was the highest (P < 0.01) for cup shaped udder (16.41 cm).Least squares means and standard errors for TD, FTL, RTL, DFT and DRT of sire, inbreeding status, parity, udder shape and age at first calving are presented in Table P > 0.05) by any of the studied factors except udder shape which affected (P < 0.01) TMY, 305d-MY and LL but not did for PMY and PR. Bowl udder produce the highest (P < 0.01) TMY and 305d-MY which were 3267.19 and 2443.01 kg and had the longest (P < 0.01) LL being 480.70 day.From Table Estimates of heritability and genetic correlation for udder and teat measurements with milk production traits are summarized in Table P < 0.05) positive correlations with milk production traits and ranged from 0.23 to 0.87. Also, UFD and URD had high (P < 0.05) positive correlations with milk production traits except that between URD and PR which were moderate positive but insignificant. The correlations between ULV, UH with milk traits were negative except those with PR which were nearly equal zero (0.03). On the other hand, the correlations between teat measurements and PR were moderate negative (P < 0.05). Also, the correlations between teat measurements with TMY, 305d-MY and LL approached zero (P > 0.05). Beside, TD, DFT and DRT teats were moderated correlated (P < 0.05) with PMY. The same trend was found for regression; udder width had the highest R2 values for all milk traits except persistency and ranged from 31.61 to 75.22 followed by URD and UFD. The regression coefficients indicated that, for each 1 cm increase in UW an increase of 110.8 kg (R2 = 70.41) in TMY and of 10.79 day (R2 = 40.26) increase in LL occurred. Low R2 values were found for ULV and UH (ranged from 0.09 to 11.95) and all teat measurements (ranged from 0.06 to 6.95) with milk production traits.Simple correlations and regressions of some milk production traits on the examined udder or teat characteristics (cm) are presented in Table 2 = 74.02, 77.43 and 42.26, respectively) and then no other variables met the level of significant. In contrast, teat measurements were entered in equation for predicting PMY and PR but with low change in R2. DRT was the first measurement entered for predicting persistency (R2 = 6.67) followed by UW with change in R2 = 4.97.Stepwise multiple regression analysis was utilized to predict milk traits from udder and teats measurements as shown in Table By investigating the QTL database for the cattle, the results revealed that there are 193,641 QTLs uncovered by 1122 different studies which were associated with 686 different economic traits. 4040 QTLs out of 193,641 were associated with udder traits , round (27.4 cm) and goaty (25.2 cm) udder shapes. Also, it was higher than 17.2 cm found in Romanian Black Spotted cows45. The hind udder depth results were much lower than 33.7 cm and 31.2 cm that reported for cup and round udder shapes of Friesian dairy cows, respectively44. The present result of udder rear depths for bowl udder shape (28.3 cm) was similar to that reported by Avarvarei45 in Romanian Black Spotted cows. The present results of teat measurements were similar to those of Deng et al.46. Milne47 revealed that morphology of the teat is recognized as part of the passive defence mechanism against intra-mammary infection, so short teats are more favourable for high milk producing cows than long teats.The calculated coefficients of variation revealed low discrepancies among cows for udder characteristics which confirm a possible genetic control for udder conformation. Every udder shape has its own specific characteristics which impose an expressive effect on udder and teat parameters. The present results on udder circumference were similar to those reported by Mona and Semaida28 who reported that the udder measurements were affected by lactations number because udder tissue might be continuous to develop up to parity six then after starts to regress thereafter. This was confirmed by Singh et al.48 who reported that all udder and teat measurements had showed increasing trends up to the 5th parity. Consistently, Bhuiyan et al.14 reported that udder length increased gradually up to lactation six. Modh et al.49 reported an increasing trend at the rate of 24.3 and 9.7% in udder length and width, respectively of Gir cows at the first two parity, but afterwards the udder length became static while udder width became static up to parity four then exhibited 15.16% increased between parity four and five, whereas udder depth was at par. However, the mean udder depth increased from 19.4 to 28.5 cm, respectively in Vrindavani cattle48 similar to the present results. Modh et al.49 reported gradual increase in length, width and depth of the udder as the number of parity increases; multiparous cows had larger volume of udder than primiparous cows. The differences observed in the present study in udder length, width and depth in different parities were statistically significant (P < 0.05). Similar findings were reported by Patel50.The effects of parity and udder shape on udder and teat measurements in the current study were similar to those reported by Tilki et al.49 who obtained FTL and RTL of Gir cows in different parities ranging from 8.74 to 9.82 and 7.97 to 8.88 cm, respectively with gradual non-significant increase in length of fore (7.56%) and rear (8.28%) teat between parities one and two. Moreover, Patel et al.51 found significant differences observed between FTL and RTL in different parities of crossbred cows ranging from 5.48 to 6.52 cm and 4.92 to 5.94 cm, respectively with gradual increase in length by advancement of parity. Similar results were reported on Holstein, Vrindavani cattle, Hariana cows, Tharparkar cows, Kankrej cows and Gir cows by many workers52. With respect to teat diameters, Modh et al.49 found that the differences observed between fore and rear teat diameters in different parities were at par and no definite trend was detected with advancement of parity. Similar results were recorded by Sharma et al.16 on Hariana and Tharparkar cows. For teats length, Antalík and Strapák53 observed a gradual increase with advancement parity order. Similar to the current results, Tilki et al.28 and Antalík and Strapák53 reported that stage of lactation did not exert any effect on the udder or teat measurements.The current FTL and RTL were smaller than those reported by Modh et al.54 who found that parity had no significant effect on lactation length and 305-day milk yield of Friesian cows. On the contrary, Mellado et al.55 found that parity had significant influence on milk yield. Also, Lee and Kim56 reported that the differences in total milk yield among parities were significant. The contradicting results may be attributed to difference in analytical models, in herd size and the age of animals. However, the relationship between udder shapes and milk yield of cattle are well established. The present results are in agreement with those of Ghosh and Prasad57 and Bhuiyan et al.14 who obtained high milk yield from cows having bowl shaped udder compared to other udder shapes in cows. Also, Prasad et al.58 found that Murrah buffaloes bowl shaped udders produced the highest average daily milk yield followed by pendulous, globular and then goaty udders. Thus raising cows with bowel shaped udder may improve the efficiency of milk production.The present parity effect on milk production traits was not significant. These results were in agreement with Atakan59 udder depth was the most unfavourably correlated with milk yield but the rear udder height and width were the most favourably related to milk yield. Also, Berry et al.60 reported mild positive genetic correlation (0.36) between udder support and milk yield. DeGroot et al.61 reported negative genetic correlations (− 0.45) between fore-udder attachment and milk yield. On the contrary, Samoré et al.62 found favourable correlation between strong fore-udder attachments and high yield of Brown Swiss data.The high positive genetic correlation of UW, UFD and URD with milk production activities were in agreement with these evaluated by various authors. Such high genetic correlations indicate that genetic selection for high milk yield will be associated with wider udders. In the current study and in previous literature for Holsteins 63. Mavrogenis et al.64, reported that the relationships between teat measures and milk production were generally low. Also, Otwinowska-Mindur et al.65 found that the genetic correlations of persistency measures with rear teat placement were rather low negative. this should be regarded as favourable, because selection for better persistency would decrease the scores for rear teat placement towards the optimum of this trait.High genetic correlations between the teat diameter and milk production indicate that genetic selection for milk yield will establish cows with substantial teat size. However, weak correlations were estimated between front teat placement, teat length, rear teat position and central ligament with milk yield 2 for udder and teat measurements with milk production traits concluded that environmental variation contributed the major part of the total variation for the milk production traits, thus management may be an effective factor in improving such traits. In addition, the heritability estimates for udder and teat measurements can change without any genetic change occurring when the environment starts contributing to more variation. In the previous studies, heritability recorded for rear udder height was − 0.7759 and 0.3161. Špehar et al.66 obtained a heritability value of 0.14 for fore udder attachment. The heritability of the teat diameter in this study was similar to that reported by Seykora and McDanel17 which was 0.44 and that of teat length was equal to Tapki and Guzey59 estimate.In this study, heritability estimates for udder and teat measurements were low to moderate. Low estimates of h67.In this study, UH and RTL having low heritability declared the importance of environmental and non-additive genetic effects on alteration of these traits. However, the traits moderate to high heritability estimates may show response for direct selection and reasonable correlation response for other traits. The magnitudes of these values indicate that a considerable proportion of the phenotypic variation occurs due to differences in genes with additive effects and that genetic gain in response to selection procedures might occur. Heritability values of udder characteristics in various studies show great variability depending on breed and housing and scoring system2 was the most suitable indicator for predicting milk traits follow by udder front and rear depth. None of the teat measurements could be used efficiently for predicting milk yield in this dataset. Deng et al.46 obtained relatively high positive correlation coefficient (0.64) between milk yield and udder length and concluded that the latter should be an important criterion for selecting dairy cows and can be used with a satisfactory precision for predicting milk yield because the regression of milk yield on udder length had the highest R2and each cm increase in udder length represent 0.22 kg extra yield of milk. Sinha et al.68 reported 0.51, 0.51 and 0.55 coefficients of determinations for the regressions of total milk yield, 305 days milk yield and peak yield, respectively on udder width and Singh et al.48 reported higher accuracy of prediction for the regression of milk yield on udder width measurement.The current UW having the highest correlation coefficients and R69. Genomic regions related to the udder and teat traits were reported in several dairy cattle38. Numerous reliable studies uncovered many QTLs and their association with udder and teat structure, as economic and significant phenotypes  were related to front teat placement, rear teat placement and rear udder width. Also, Marete et al.72 reported that 10 candidate genes were uncovered for their associations with udder traits, including RREB1, FGF2, FGFR-2, ESR-1, IQGAP-3, GLI-2, PGR, BTRC, TGFBR-2 and PRLR, in French dairy cattle. Furthermore, 18 candidate SNPs within STXBP-6 and SLF-1 genes were related to five udder traits in the Chinese Holstein breed38. Meanwhile, several SNPs within LGALS-2, GCLC and ADGRB-3 genes were related to udder depth trait in Holstein cattle73.Concerning the genetic background of udder characteristics, the heritability of udder and teat measurements/characteristics is moderate 0.23–0.45), which in turn facilitates the genetic improvement programmes3–0.45, w74, mastitis resistance75 and longevity76. Thus, there is a great benefit to considering and investigating these traits side by side with their genetic background in future studies.On the other side, udder traits are related to sustainable milk productionResult of the present study shows that parity had significant effect on udder and teat characteristics. Udder shape has a considerable relationship and affects milk yield and lactation length. The bowel udders shape produces high milk yield and associate with long lactation length compared to other udder shapes, therefore, should be considered while selecting Friesian cows. The genetic correlation of udder, teat measurements with milk yield indicated a correlated response; therefore, selection to improve teat diameter and udder width should increase milk yield.All udder and teat characteristics could be included in a selection index criterion (except UH and RTL) to improve the total merit of lactating cows directly or through correlated response with other traits. Also, udder width and udder rear depth can be used as a good indicator for predicting milk yield and lactation length. Finally, the detected QTLs, candidate genes and significant SNPs are potential tools to improve milk productivity side by side with the udder phenotypic investigations.


Moreover, an association was identified between a reduction in tumor mass and the highest tertile of plasma bacterial DNA. Our findings indicate that individuals with CRC displayed a higher plasma bacterial DNA load compared to healthy controls. This observation lends support to the theory of heightened bacterial migration from the gastrointestinal tract to the bloodstream in CRC. Furthermore, our results establish a link between this phenomenon and the size of the tumor mass.The gut microbiota has gained increasing attention in recent years due to its significant impact on colorectal cancer (CRC) development and progression. The recent detection of bacterial DNA load in plasma holds promise as a potential non-invasive approach for early cancer detection. The aim of this study was to examine the quantity of bacterial DNA present in the plasma of 50 patients who have CRC in comparison to 40 neoplastic disease-free patients, as well as to determine if there is a correlation between the amount of plasma bacterial DNA and various clinical parameters. Plasma bacterial DNA levels were found to be elevated in the CRC group compared to the control group. As it emerged from the logistic analysis (adjusted for age and gender), these levels were strongly associated with the risk of CRC (OR = 1.02,  Colorectal cancer (CRC) is the third most common cancer diagnosed worldwide, and patients diagnosed with distant-stage CRC have low 5-year survival rates 15%), compared with 90% for early stages 5%, compa,3. ThereFusobacterium nucleatum (F. nucleatum) is highly enriched in CRC tissues and many studies have reported that F. nucleatum promotes tumor development by creating a more favorable microenvironment for cancer growth [Bacteroides fragilis and the genus Porphyromonas have been also associated with an increased risk of CRC [Measuring cancer-related bacterial DNA in plasma cell-free DNA (cfDNA) might represent an accurate and non-invasive approach for early cancer detection, as demonstrated in previous studies ,15. Somer growth ,17,18,19k of CRC , while ak of CRC .Previous results conducted by our research group in a large European population revealed that circulating bacterial DNA load was associated with fatty acid (FFA) levels and leukocyte count,  which reThe present study included a cohort of 90 subjects in the age range 30–91 years, enrolled at the National Institute of Gastroenterology “S. de Bellis” , from March 2017 to November 2021. The exclusion criteria were the following: HIV, HBV and HCV seropositivity; use of glucocorticoids; inability to provide informed consent; presence of acute illness or infections within fourteen days preceding blood collection.®  and were processed for the separation of plasma. Briefly, blood venous samples collected from all subjects were kept and then centrifuged for 15 min at 2000× g at room temperature. The plasmas were divided in 500 µL aliquots, transferred in cryovials and stored at −80 °C in the Biobank of the IRCCS of S. de Bellis .Specifically, 50 blood samples from patients with CRC  and 40 blood samples from healthy subjects  were collected into 3 mL K2 EDTA VacutainerThe patients have read and signed the informed consent approved by the ethics committee of the IRCCS “Giovanni Paolo II”—Oncological Institute (Bari) with protocol number (Prot. no. 379/C.E. of 16 September 2020).Leukocyte, neutrophil and lymphocyte counts were determined using Coulter Hematology analyzer . The SIRI index was calculated as previously reported . FastingDNA was extracted from 200 µL of plasma biobanked samples using plasma/serum circulating DNA mini kit  according to the manufacturer’s instructions. Highly sensitive and specific universal primers targeting the V3-V4 hypervariable region of the bacterial 16S rDNA were used in real-time qPCR reactions to quantify the 16S rRNA gene levels in DNA samples. The PCR mixture (20 µL) consisted of 20 ng of DNA, SensiFAST SYBR Hi-ROX Mix 1X  and 0.4 µM of the following primers: Forward 5′-TCCTACGGGAGGCAGCAGT-3′ and Reverse 5′-GGACTACCAGGGTATCTAATCCTGTT-3′. The thermal profile used for the reaction included a heat activation of the enzyme at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 60 s, followed by melt analysis ramping at 60–95 °C. All measurements were taken in the log phase of amplification. Standard curves obtained using a 10-fold dilution series of bacterial DNA standards  ranging from 2 ng to 200 fg were routinely run with each sample set and compared with previous standard curves to check for consistency between runs. Amplicon quality was ascertained via melting curves. Amplifications of samples and standard dilutions were performed in triplicate on the StepOne Real-Time PCR System . Bacterial DNA levels were expressed as pg per mL of whole blood. A series of controls both in silico and in vitro were performed to exclude artifacts from sample manipulation, reagent contamination and non-specific amplifications, the primers were checked for possible cross-hybridization with genes from eukaryotic and mitochondrial genomes using the database similarity search program, and separate working areas were used for real-time PCR mix preparation, template addition and for performing the PCR reactions ,26. NegaPatient characteristics are reported as mean and standard deviation (M ± SD) for continuous variables, and as frequency and percentage (%) for categorical variables. Shapiro–Wilk test was used to test the normality of variables distribution. To test the association between the independent groups (CRC vs. control group), the chi-squared or Fisher’s test was used for categorical variables, where necessary, and the Wilcoxon rank Mann–Whitney test was used for continuous variables.A logistic regression model was used to evaluate the associations of status (CRC vs. control) on the single variables examined, with 95% Confidence Interval (95% C.I.), and covariate as age and gender were used to adjust the models. Dunn’s test of multiple comparisons was used to compare MTD in DNA tertile group.The Spearman rank correlation coefficient was employed to assess the strength and direction of the association between the two variables under examination .To test the null hypothesis of non-association, the two-tailed probability level was set at 0.05. The analyses were conducted using StataCorp. 2023. Stata Statistical Software: Release 18. College Station, TX, USA: StataCorp LLC., and RStudio (“Prairie Trillium” Release) was used for the plots.p < 0.001).As shown in p < 0.0001). Noteworthy, significant differences were observed between the two groups, in terms of Body Mass Index (BMI)  and smoking behavior (p = 0.01). The CRC group displayed a higher prevalence of conditions such as hypertension, diabetes and comorbidities (p < 0.05). There was a significant difference in blood parameters  among CRC patients compared to the control group. Plasma bacterial DNA appeared to be higher in CRC than controls . While tumor variables, present only in the CRC group, were recorded to describe the disease severity.Patients with CRC were older than controls . Hypertension, comorbidities and leukocyte subsets and the SIRI index were significantly associated with the risk of CRC in both logistic models. An association was found between a decreased tumor mass and the highest tertile of plasma bacterial DNA  (p = 0.01) and SIRI index .Plasma bacterial DNA both in the unadjusted and in the adjusted model (for age and gender) was strongly associated with the disease  . Correla = 0.04) . In the No correlation with tumor markers and other laboratory parameters was found in CRC patients .Fusobacterium, Firmicutes and Lactococcus, and the under-representation of Proteobacteria, Escherichia-Shigella and Pseudomonas in cancerous tissues [In the last five years, numerous studies have demonstrated the unequivocal presence in the bloodstream of a circulating microbial DNA, both in physiological conditions and in several pathologies, such as type 2 diabetes, cancer as well as metabolic, neurodegenerative and cardiovascular diseases ,11,12,13 tissues . A furth tissues  analyzedVarious risk factors may induce gut microbiota dysbiosis, including host genetics, nutrition , smokingThe most relevant aspect of this study is the association between plasma bacterial DNA levels and tumor mass dimension. Since tumor size has been considered an independent prognostic parameter for subjects with colorectal cancer, having observed that the higher bacterial DNA levels correlate with a lower tumor mass led us to hypothesize that there is a critical phase occurring in the early stages of tumor development in which the microbial translocation in the plasma is massive. As the tumor mass expands, the bacterial population in the blood could, then, undergo a clearance by the immune system as described by some authors ,43. HoweSince systemic inflammation response index (SIRI) seems to have a prognostic role in CRC patients and a high SIRI index is associated with lower microbial richness and diversity , we analWe found a lack of association between elevated plasma bacterial DNA and lymph node metastases or tumor stage  that couThe high prevalence of CRC causes the identification of a set of clinically useful blood-based screening tools to be challenging. In this study, we provide, for the first time, the evidence that plasma bacterial DNA levels together with the anthropometric and hematological parameters, including BMI, lymphocyte and neutrophil counts, as well as the presence of comorbidities could serve as a potential biomarker for efficient CRC screening. Moreover, plasma bacterial DNA load, by correlating with the tumor mass, could be used to detect the early stages of tumor development. Our study presents some limitations. These include a relatively small sample size, the absence of detailed bacterial species characterization among CRC patients and the omission of an analysis regarding the potential influence of different types of therapy on dysbiosis and plasma bacterial DNA levels. Moreover, the observed heterogeneity within this group can be attributed to the limited sample size, which is reflective of real-world constraints. This bias can certainly be addressed and mitigated by including a larger cohort of patients in future research articles.Our results reveal that CRC patients exhibited a greater plasma bacterial DNA load in comparison to their healthy counterparts, thus supporting the theory of an increased bacterial migration from the gastrointestinal tract to the bloodstream and demonstrating an association with the size of the tumor mass. Future research should aim to validate these findings in larger and more diverse cohorts. Expanding the sample size can enhance the robustness of the association between plasma bacterial DNA levels and CRC risk. Moreover, prospective studies should be conducted to assess the predictive value of elevated plasma bacterial DNA levels for the development of CRC. Investigating the bacterial species linked to the onset and progression of CRC may prove valuable in developing personalized treatment strategies for CRC patients. By identifying specific bacterial signatures associated with different stages of CRC, clinicians could tailor treatments to target the microbiome and complement existing therapies, potentially improving patient outcomes and reducing side effects.


Ne), genetic diversity, genetic distances, and population differentiation and structure. The LD pattern represented by average r2, decreased to <0.2 when the inter-marker interval reached larger than 350 kb and 650 kb for CCFAR and MFF, respectively, and suggested at least 7,700 and 4,200 single nucleotide polymorphisms (SNPs) be used to obtain adequate accuracy for genomic selection programs in CCFAR and MFF respectively. The Ne for five generations ago was estimated to be 76 and 91 respectively. Our results from genetic distance and diversity analyses showed that American mink of the various color types had a close genetic relationship and low genetic diversity, with most of the genetic variation occurring within rather than between color types. Three ancestral genetic groups was considered the most appropriate number to delineate the genetic structure of these populations. Black (in both CCFAR and MFF) and pastel color types had their own ancestral clusters, while demi, mahogany, and stardust color types were admixed with the three ancestral genetic groups. This study provided essential information to utilize the first medium-density SNP panel for American mink in their genomic studies.Understanding the genetic structure of the target population is critically important to develop an efficient genomic selection program in domestic animals. In this study, 2,973 American mink of six color types from two farms , Truro, NS and Millbank Fur Farm (MFF), Rockwood, ON) were genotyped with the Affymetrix Mink 70K panel to compute their linkage disequilibrium (LD) patterns, effective population size ( Neogale vison) is a semiaquatic and carnivorous mammal that belongs to the weasel (Mustelidae) family (American mink  family . It is n) family . With th) family . However) family , swine  family , and pou) family , to imprNe) are two important parameters for revealing genetic structure of target population. The LD is defined as the non-random association of alleles at two or more loci  markers. Microsatellite loci were used to investigate the genetic structures of wild-caught American mink in Japan , Sweden n Canada . Moreoven Canada . HoweverNe of farm American mink in Canada, 2) explore the genetic distance and genetic diversity among various color types of American mink, and 3) reveal the genetic structure and admixture pattern of farm American mink in Canada.Investigation of the genetic structure of American mink using genotypic data from a medium-density SNP panel will benefit the future use of this genotyping panel for use in genomic selection, as well as other genomic studies, such as quantitative trait locus mapping, identification of signatures of selection, and GWAS. Meanwhile, one critical factor affecting the accuracy of estimating population genetic diversity parameters is the sample size . The samThis study was approved by the Dalhousie University Animal Care and Use Committee (certification#: 2018-009 and 2019-012). All the mink were raised based on the Code of Practice for the Care and Handling of Farmed Mink guidelines from the Canada Mink Breeders Association .n = 1,411) at Dalhousie University, Faculty of Agriculture  and Millbank Fur Farm  at Rockwood . Mink from CCFAR included five color types: black , demi , mahogany , pastel , and stardust . The colors of the studied mink were identified and assigned to them at their weaning age by experienced technicians at CCFAR. All individuals from MFF were Black color type . There was no migration of mink between the two farms. There was no regular mating system on both farms, and breeders were selected based on their phenotypic performances without considering the color types.The individuals used in this study were from two farms, including the Canadian Center for Fur Animal Research , according to the manufacturer’s instructions. The quantity and quality of DNA were measured with a NanoDrop ND-1000 spectrophotometer . The 260/280 nm readings for all samples ranged from 1.8 to 2.0. All samples were diluted to a final concentration of 500 ng, checked for DNA quality, and finally genotyped by Axiom Affymetrix Mink 70K panel  .−5), and individuals with a call rate lower than 90%. Overall, 2,973 genotyped animals with 24,161 SNPs remained for the following analyses.Prior to analyses of the genotyping data, animals and SNPs were excluded from the dataset based on the following criteria using PLINK software : SNPs haThe average minor allele frequency (MAF) and observed heterozygosity were estimated for each color type and whole CCFAR population using SNP1101 software . The nucr2) was measured as proposed by ij is the frequency of the two-marker haplotype , and Pi and Pj are the frequencies of allele i at locus 1 and allele j at locus 2, respectively  after transforming data using a principal component analysis (PCA). The most suitable number of clusters has the lowest associated Bayesian Information Criterion (BIC). An α-score optimization was used to determine the number of principal components to retain. Additionally, an unsupervised analysis in ADMIXTURE version 1.3.0  method using the “adegenet” package of R . The numon 1.3.0  was applThe MAF, observed heterozygosity, and nucleotide diversity for each SNP, color type and whole CCFAR population are present in r2 between adjacent SNPs on all chromosomes for six color types of American mink and the whole CCFAR American mink population are presented in r2 between adjacent SNPs among various color types ranged from 0.373 ± 0.402 (CPA) to 0.406 ± 0.408 (MBL). Compared with the MFF population (MBL), the average r2 between adjacent SNPs for the whole CCFAR population (0.399 ± 0.404) was lower  and consecutive bins  in six color types mink is presented in r2 among all color types at the same inter-marker distance, while CDE and CPA had the two lowest average r2 among all color types. CDE reached an average r2 < 0.2 with the shortest inter-marker distance (around 325 kb) among all color types, while CST reached an average r2 < 0.2 with the longest inter-marker distance (around 850 kb) among all color types. Both CMA and CPA reached an average r2 < 0.2 at the inter-marker distance of approximately 350 kb. CBL and MBL reached an average r2 < 0.2 at the inter-marker distance of approximately 475 kb and 650 kb, respectively. The average r2 of the whole CCFAR population was <0.2 at the inter-marker distance of 350 kb. The most rapid LD decays for all color types were observed when the average inter-marker distances increased from 50 to 150 kb, and CDE had the most rapid reduction of LD in this interval  from five to 250 generations ago, and the estimates of Ne are shown in Ne, five generations ago, of CBL, CDE, CMA, CPA, CST, and MBL was 58, 76, 80, 60, 24, and 91, respectively. For the whole CCFAR population, the Ne was 76 at five generations ago. The maximum Ne was observed 250 generations ago for all color types, where CDE had the highest Ne of 384 and CST had the lowest Ne of 276. CDE had the highest Ne, and CST had the lowest Ne from 50 to 250 generations ago. However, in more recent generations, from five to ten generations ago, MBL was observed to have the highest Ne, and CST was found to have the lowest Ne. The decline of Ne was more rapid from 5 to 50 generations ago for all color types in CCFAR and from 5 to 100 generations ago for the MFF population. In the meantime, in more recent generations (5–50 generations ago), the Ne of the CCFAR population decreased more rapidly than the MFF population (The pulation .Weir and Cockerham’s Fst and Nei’s genetic distances among six color types are shown in p < 0.05) but only represented 4.1% of the total molecular variation in the populations. The variation among farms was estimated to represent 4.3% of the total molecular variation in the populations but was not significant (p > 0.05).The results from AMOVA for various color types are shown in y-axis, and most MBL individuals were grouped into those clusters , and the other two clusters were dominant in the CCFAR population. The CBL (average of 77.51% on ancestry fractions) and CPA (average of 79.13% on ancestry fractions) became distinct clusters within the CCFAR population and the two main genetic compositions in the CCFAR population. In the meantime, CDE and CMA were admixed with the three clusters and seemed to share a similar admixture pattern. When K = 4 and 6, except for the CPA color type, where one cluster seemed like the dominant cluster in this color type, all other color types were admixed with at least two clusters. In the meantime, the CCFAR population showed a higher level of admixture than the MFF population. When K = 40 and 75, no obvious distinction in ADMIXTURE among various color types was found, and all color types were admixed with several clusters  and 12,000 (2.68 Gb/225 kb) SNPs are necessary to conduct GWAS in CCFAR and MFF populations, respectively. It has been noted that more markers are needed to perform adequate genomic studies in admixed populations  for CCFAR and a lower Ne at five generations ago (50) for MFF using SNP information extracted from the WGS data of 100 mink. The estimates of the current study indicated that the Ne declined more rapidly from 5 to 50 generations ago for the CCFAR and from 5 to 100 generations ago for the MFF population, which coincided with the time when the farms were established. The CCFAR was established about 40 years ago (1984), and the MFF was founded over 90 years ago (1930). These trends were different from those estimated by using WGS data (Ne was more rapid between 150 and 200 generations ago. The different estimations of LD patterns among studies are the cause leading to the different Ne estimates. The Ne of the CCFAR population decreased more rapidly than the MFF population in more recent generations (5–50 generations ago). This may be related to different breeding managements and strategies, population genetic backgrounds, and populations sizes (MFF has larger population size than CCFAR) in these two farms.In this study, the ite loci . On Swed markers . The Ne GBS data . CompareWGS data  and GBS WGS data , where tBoth Fst (less than 0.1) and Nei’s genetic distances (less than 0.06) among various color types were low, which indicated the low genetic differentiation among various color types. This was in agreement with the AMOVA results, where the variation within color types explained 91.6% of total molecular variation, while the variation among color types only explained 4.1% of the total molecular variation in the populations. Compared with other color types, CST had the farthest genetic distances  with CDE, CPA, CMA, and MBL color types. This was in agreement with the result from the phylogenetic tree, which separated CST into a separate cluster from other color types. The Fst and Nei’s genetic distances among various color types were estimated in the range of 0.015–0.124 and 0.013 to 0.065, respectively, using WGS data . They wey-axis of the DAPC scatterplot of the first two linear discriminants  and a relatively single breeder source in MFF. Meanwhile, MBL and CBL were clearly identified with a distinct ancestral population suggesting that these two black color types derive from different ancestral populations and color type might not be a reliable indicator to differentiate American mink. Within the CCFAR population, CBL and CPA had their own clusters, and CDE, CMA, and CST showed noticeable admixtures of these two clusters. Many color types in American mink are exclusively line-bred because many color types are recessive to the standard brown color type, and the rest are blended  the  blended ; 3) CDE Ne decay , 2; and Ne decay . These rNe decay , which wNe decay . Similare groups .Genetic structure and admixture pattern analyses conducted in this study did not detect clear genetic distinctions among the mink of various color types. Two potential reasons could lead to these results. The studied individuals were sampled from only two farms, which might make the samples not the ideal sample structures to reveal the population structures. Thus, future studies should consider including animals of various color types from wild and more geographically distributed farms. In the meantime, it has been noted that a larger number of markers may be needed to resolve population genomics studies when the genetic distance (Fst) between the populations is low . Thus, fIn this study, 2,973 animals from two farms and their genotypes obtained from the first developed medium-density SNP panel for American mink were used to investigate their LD patterns and genetic structure in various color types. The estimated LD patterns suggested that 7,700 and 4,200 SNPs are the minimum marker densities to conduct genomic selection programs in CCFAR and MFF populations, respectively. The results from genetic distances and diversity analyses indicated genetic differentiation among various color types was low, and most of the genetic variation occurred within color types rather than between color types. Three ancestral genetic compositions were considered the most appropriate number of ancestral genetic compositions to delineate the populations’ genetic structure. The black (both CCFAR and MFF) and pastel color types seemed to have their own ancestral clusters, and demi, mahogany, and stardust were admixed with the three ancestral genetic compositions. Additionally, mink in demi and mahogany color types seemed to have a similar admixture pattern, but further study is needed. The genetic structure and admixture pattern of mink of various color types and within the same color type were not clearly identified in this study. Thus, future studies with samples from wider geographically distributed locations and higher marker density are needed to differentiate the mink within the same color type.This study provided useful information for conducting genomic evaluations in the mink industry using genotypes from the medium-density SNP panel. The mink industry faces several challenges caused by the COVID-19 pandemic, industry downturn, and decreasing market demand. Improving production efficiency through advanced genomic approaches could help the mink industry meet these challenges. The LD patterns and genetic structures obtained from the first SNP panel for American mink would provide the essential information to implement the SNP panel in genomic studies of American mink.