Loneliness is a serious public health issue, and its burden is increasing in many countries. Loneliness affects social, physical, and mental health, and it is associated with multimorbidity and premature mortality. In addition to social interventions, a range of digital technology interventions (DTIs) are being used to tackle loneliness. However, there is limited evidence on the effectiveness of DTIs in reducing loneliness, especially in adults. The effectiveness of DTIs in reducing loneliness needs to be systematically assessed.The objective of this study is to assess the effectiveness of DTIs in reducing loneliness in older adults.We conducted electronic searches in PubMed, MEDLINE, CINAHL, Embase, and Web of Science for empirical studies published in English from January 1, 2010, to July 31, 2019. The study selection criteria included interventional studies that used any type of DTIs to reduce loneliness in adults (aged ≥18 years) with a minimum intervention duration of 3 months and follow-up measurements at least 3 months after the intervention. Two researchers independently screened articles and extracted data using the PICO  framework. The primary outcome measure was loneliness. Loneliness scores in both the intervention and control groups at baseline and at follow-up at 3, 4, 6, and 12 months after the intervention were extracted. Data were analyzed via narrative synthesis and meta-analysis using RevMan (The Cochrane Collaboration) software.P=.92), 4 months , and 6 months . The quality of evidence was very low to moderate in these trials.A total of 6 studies were selected from 4939 screened articles. These studies included 1 before and after study and 5 clinical trials . All of these studies enrolled a total of 646 participants  with an average age of 73-78 years (SD 6-11). Five clinical trials were included in the meta-analysis, and by using the random effects model, standardized mean differences (SMDs) were calculated for each trial and pooled across studies at the 3-, 4-, and 6-month follow-ups. The overall effect estimates showed no statistically significant difference in the effectiveness of DTIs compared with that of usual care or non-DTIs at follow-up at 3 months (SMD 0.02; 95% CI −0.36 to 0.40; Our meta-analysis shows no evidence supporting the effectiveness of DTIs in reducing loneliness in older adults. Future research may consider randomized controlled trials with larger sample sizes and longer durations for both the interventions and follow-ups.RR2-10.1136/bmjopen-2019-032455 Loneliness is a multifaceted public health problem . The burLoneliness refers to an individual’s subjective feelings of a perceived discrepancy between actual and desired social relationships ,15. AlthLoneliness is being addressed through a range of social  and techSeveral published reviews have reported that digital technology interventions (DTIs) are effective in reducing loneliness -34. HoweThere is limited evidence on the effectiveness of DTIs for loneliness , and theThe primary objective of this study is to assess the effectiveness of DTIs in reducing loneliness in adults. The secondary objective is to identify DTIs that are used to reduce loneliness in adults.The main research question was “Are DTIs effective for reducing loneliness in adults?” The secondary question was “What DTIs are used for reducing loneliness in adults?”The main outcome measure was loneliness. We extracted data on loneliness measured at both the baseline (before the intervention) and follow-ups (at least 3 months after the intervention) for the intervention groups and control groups, if any.We undertook a systematic review and meta-analysis as suggested in the Cochrane Methods for Systematic Reviews of Interventions . We haveWe registered this systematic review and meta-analysis with the PROSPERO database on June 10, 2019 (registration ID: CRD42019131524) , and we A patient and public manager affiliated with our research center reviewed the study protocol and provided suggestions that were incorporated into the protocol. We had no access to any patient diagnosed with loneliness; therefore, we could not include any patients or members of the public in the design and conduct of the study. However, the findings of this study will be disseminated as an open access publication that will be freely available to patients and everyone else globally.We define the term DTI as an intervention that applies digital technology, that is, the technology, equipment, and apps that process information in the form of numeric codes, usually a binary code .We selected studies that met our predefined eligibility criteria . Study dloneliness, lonely, isolation, aloneness, disconnect*, solitude, singleness*, lonesomeness, solitariness, and remoteness) and intervention or technology , as reported in our published protocol [We electronically searched PubMed, MEDLINE, CINAHL, Embase, and Web of Science and covered the publication period from January 1, 2010, to July 31, 2019. We used an a priori list of keywords prepared in our preliminary literature searches . The keyprotocol .subject headings such as MeSH major terms in PubMed or equivalent terms in other databases  reviewed these articles, and his recommendations to either include or exclude an article were final.Finally, 92 articles were excluded, and the remaining 6 articles were included in the data extraction . All theFor data collection, we used an a priori data extraction template  and 2, wSGSS and DN independently extracted data from all included studies (n=6) using the data extraction template  and 2 anWe report both a narrative synthesis (narrative summary) and a statistical (quantitative) synthesis  of our review, as suggested for reporting of a systematic review on effectiveness . In the In the meta-analysis, we have included 5 studies and pooled extracted data on loneliness measured by continuous loneliness scales, that is, the University of California, Los Angeles (UCLA) loneliness scale ,51 in 4 d and includes an adjustment for small sample size bias [In meta-analysis, the standardized mean difference (SMD) as a summary statistic for reporting continuous data has been suggested for studies that assess the same outcome but use different scales to measure the outcome . In RevMize bias . More imize bias .In our review, the main outcome, that is, loneliness, was measured using different loneliness scales, which included the UCLA loneliness scale (score range 20-80) ,51 and tThe Cochrane guidelines for systematic reviews and meta-analysis  suggest We extracted data from 6 studies, which included 5 clinical trials ,50,53,54We calculated the SMDs from the extracted data, that is, loneliness mean scores with SD and sample sizes in the intervention and control groups at follow-up measurements at 3 months and beyond. For conducting meta-analysis, we used the Cochrane Review Manager (RevMan) software, version 5.3.5 . In the We assessed the quality of research by applying the GRADE  approach .We assessed the risk of bias by focusing on 5 domains: the evaluation of sequence generation, allocation concealment, blinding (outcome assessors), incomplete data, selective outcome reporting, and assessing other biases using the Cochrane guidelines . In a meρ<0.10 [2 statistics to determine the magnitude of heterogeneity  between studies [2 values of 25%, 50%, and 75% as low, moderate, and high heterogeneity between studies, respectively [We checked heterogeneity, that is, variation in study outcomes or intervention effect sizes between studies, by the Cochran Q test with a significance level of ρ<0.10 ,73 becauρ<0.10 . We calc studies . We consWe report the findings of meta-analyses using SMDs with 95% CIs as a statistical summary, with the forest plots .Findings about the characteristics of the studies, including the study designs, settings, participants, interventions, comparators, sample sizes, participant attrition, and data collection methods or tools used, are presented in Searches of PubMed, MEDLINE, CINAHL, Embase, and Web of Science generated a total of 4939 articles , of whicThe total number of participants enrolled in all 6 included studies was 646 . Studies varied in total sample sizes , and the sample sizes of the intervention and control groups also varied at both the baseline and follow-up measurements across the studies . The attParticipants’ average age was between 73 and 78 years (SD 6-11). Total enrolled participants included 66.1% (427/646) women and 23.8% 154/646) men, whereas for 10.1% (65/646) of participants, no information about their gender was available. Studies varied in the proportion of male and female participants . Only 2 studies reported on participants’ ethnicity—White (54%) and non-Whites (46%) in the US study  and most/646 men,A total of 4 studies were RCTs ,50,53,54, and Sweden [A total of 4 studies were conducted in developed countries, namely, the Netherlands , the Unid Sweden . Two stud Sweden  and Soutd Sweden .domiciliary care), or receiving care in residential care homes [The settings included living in independent housing in the community ; living re homes , residenre homes , nursingre homes , and oldre homes .Participants were selected by random sampling in 66.7% (4/6) of studies ,50,53,54DTIs included social internet-based activities, that is, social activities via social websites , videocoThe duration of the intervention was 3 months in 4 studies ,50,53,54The loneliness measurement tools used were the UCLA loneliness scale ,51, whicMeta-analysis section.Narrative synthesis showed that there was a reduction in loneliness in the intervention groups at the follow-ups compared with baseline . A statiWe conducted 3 meta-analyses, 1 each for follow-up measurements at 3, 4, and 6 months, involving 3, 2, and 2 studies, respectively.P=.92). The heterogeneity between studies was not statistically significant . There was a statistically significant high heterogeneity between studies . There was moderate heterogeneity between studies, but it was not statistically significant  as well as a meta-analysis  of the findings. The narrative summary of 6 studies included in our review showed a reduction in loneliness in the intervention groups at follow-up compared with baseline . HoweverOur meta-analysis also revealed that CIs of the summary effects of 2 studies, that is, the studies by Larsson et al  and TsaiOverall, the findings of our meta-analysis showed no evidence supporting the effectiveness of DTIs in reducing loneliness in older adults.The quality of evidence of the included studies was very low to moderate , and theLoneliness is influenced by culture -79, gendThere are limited published meta-analyses on technological interventions for tackling loneliness, and a few existing studies have covered literature published up to 2009  and 2011A meta-analysis by Masi et al  also repInterestingly, our findings provide new insights about DTIs and loneliness. Our meta-analysis showed no statistically significant reduction in loneliness in the intervention groups compared with the control groups at the 3 -, 4-, and 6-month follow-ups. Thus, our findings show no evidence supporting the effectiveness of DTIs in reducing loneliness in older adults, which goes beyond the findings of a recent Cochrane review that reported no evidence of video calls being effective in reducing loneliness in older adults .In addition, our findings refute and contradict a commonly held view that digital technology can solve the problem of loneliness, especially in older people. Nonetheless, digital technologies provide tools and means that facilitate social connection , which mNonetheless, a review has reported that some nontechnological interventions are effective in reducing loneliness in older people , but theOur study had some limitations: the inclusion of only 6 studies with heterogeneous sets of results and the minimum intervention duration of 3 months, which could have resulted in the inclusion of a small number of studies and possible exclusion of potential studies that would have provided useful evidence.In addition, we could not conduct subgroup and meta-regression analyses due to the very limited number of studies (n=5) in the meta-analysis and lack of data on loneliness by participants’ demographic characteristics. In addition, our study might be narrow because we excluded some studies ,85-91, wP=.03; Moreover, another limitation of our review could be the use of a meta-analysis based only on follow-up data. For example, a study by Tsai et al  in the 3As recommendations for future research, we suggest that researchers involved in trials agree on a common measure of loneliness and consider reporting of results in a standardized way, which will allow pooling of baseline-adjusted estimates of the treatment effect rather than differences in follow-up means.Our meta-analysis showed no evidence supporting the effectiveness of DTIs in reducing loneliness in older adults. Therefore, there is a need for further research involving RCTs  with lar


Camellia sinensis) has anti-inflammatory and antioxidant properties and a protective effect against wrinkles, sunburn and UV damages on the skin. Thus, we aimed to evaluate the effect of white tea extract on the healing process of skin wounds in rats.White tea  including control group, Eucerin-treated group, white tea 5% ointment (Eucerin) treated group, gel-treated group, white tea 5% gel treated group. Treatment was begun on day 1 and repeated every day at the same time until day 15. Pathologic samples were taken on days 4, 7 and 15 for histopathological examinations. Kruskal-Wallis test was used to analyze data by SPSS. Statistical significance was defined as P<0.05). On day 7, reepithelisation and granulation tissue of control group were more than white tea 5% ointment-treated and its inflammation was less than others (P<0.05). Neo-vascularization of white tea 5% ointment-treated group was more than control group on days 4 and 15 (P<0.05). On day 4, intact mast cells of control group were more than white tea treated groups (P<0.05). Degranulated mast cells of white tea 5% gel treated group was significantly (P<0.05) more than control group on days 4 and 15.Wound closure rate of control group was more than other groups on day 4 (Five percent white tea extract could not help the skin wound healing process. Therefore, wounds by causing a disturbance in skin anatomy and function may lead to complications such as infection and even shock 3, 4. Because of the importance of healthy skin, wounds prevalence and the costs of skin wound treatment, it is essential to find a low-cost and effective treatment for skin wounds 2,5,6.Skin protects our bodies against dehydration and foreign pathogens influx and maintains body homeostasis and temperature 7-9. Thus, the wound-healing effects of some medicinal plants have been studied and proven 4.Chemical drugs are expensive and have side effects, while, medicinal plants are safe and cheap Lemon, Aloe vera, Jojoba, and Ginseng were used for wound healing and their common feature was the production of flavonoid compounds with phenolic structures 10. Flavonoids and polyphenols have anti-oxidative effect 11.The extracts of some medicinal plants such as 12. Overproduction of ROS is cytotoxic and delays wound healing process. Therefore, one way to improve wound healing is reducing ROS 5.In a normal physiological state, production and neutralization of reactive species are in balance Camellia is a member of the Theaceae family 13. Tea (C. sinensis L) has various beneficial effects including antioxidant, anti-diabetic, neuroprotective, hypocholesterolemic, antimicrobial and antifungal activities 14,15. It also prevents cancers and heart disease and protects our body from Cd and Pb accumulation 16. C. sinensis water extract has anti-wrinkle effects 17.The genus 14,15,18. There are polyphenol oxidase and other oxidative enzymes in the leaves of tea; thus, during the fermentation process from white tea to black tea, the polyphenols of C. sinensis’ leaves become more and more oxidized 14,18. Therefore, white tea has the most antioxidants among all types of teas 15. The antioxidant property of tea seems to be due to tea flavonoids and polyphenols 17. Some of the features of polyphenols are antioxidant, antineoplastic and anti-inflammatory effects 15, 19. Moreover, they are good for skin health 19.Different types of tea are classified based on their formation process. White and green tea are non-fermented, red tea is semi-fermented and black tea is fermented 20. They are anti-oxidants that improve wound healing by increasing collagen volume and keratinocytes reproduction 21. The catechins of white tea are catechin (C), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC) 1,21, more anti-oxidants in white tea in comparison with green tea and the positive effects of antioxidants and antimicrobial agents on the wound healing process and all the properties of white tea, we aimed to evaluate the effect of white tea extract on the healing process of excisional skin wounds in rats 2,14,15,18.Considering similarities between green tea and white tea, and benefits of green tea ethanolic extract on the surgical and burn wounds After obtaining approval from Ethics Committee of Shahrekord University of Medical Sciences, this experiment was carried out in the Research Center of Shahrekord University of Medical Sciences in 2019.Preparation of white tea extract21.The Maceration method was used for preparing the extract. Dried leaves of white tea  were pulverized by a grinder machine. A mixture of 100 gr of white tea powder and 1 L of ethanol 70%  was made in an Erlenmeyer and kept for 48 h in the laboratory temperature. Subsequently, all the materials were filtered by filter paper, evaporated by a rotary and dried by a heater The powder of extract was uniformly mixed with Eucerin  to prepare Eucerin-based 5% ointment. A gel containing 5% extract of white tea was produced, too.AnimalsHealthy male Wistar rats  were housed in the same situation for light, food, and water. A standard glass cage was allocated to each rat. After acclimatization to the environment of the laboratory, 105 rats were randomly divided into five groups containing 21 rats each: control group, vehicle (Eucerin)-treated group, white tea 5% ointment (Eucerin)-treated group, vehicle (gel)-treated group and white tea 5% gel-treated group.Surgical wounds21, 22.On day 0, all the rats were anesthetized with an intramuscular injection of 20 mg/kg ketamine 10%  and 2 mg/kg xylazine 2% . Then, after the disinfection of the skin of dorsal surface of their neck by betadine 10%  and shaving the hair, a full-thickness circular excisional skin wound was created with 1 cm diameter on the dorsal surface of the neck by a sterile scissor TreatmentsAll the rats were treated every day for 15 d and the treatment began on day 1 which means one day after the surgery. Every day, all the wounds of all rats were washed by normal saline serum  and after that, all of them, except the control group, was given a layer of their local treatment which covered the wound completely. Therefore, the wounds of vehicle (Eucerin)-treated group were treated with the Eucerin vehicle, the wounds of white tea 5% ointment (Eucerin)-treated group with white tea 5% ointment, the wounds of vehicle (gel)-treated group with vehicle gel and wounds of white tea 5% gel treated group with gel 5% of white tea.Data collectingSeven rats from each group were randomly selected on days 4, 7 and 15. The surface of the remaining wound of each rat was drawn on a transparent paper and with the aid of AutoCAD software, the area of the remaining wound was measured. The following equation was used for wound closure rate calculation.Wound closure rate %= [(area at day 0 – area at each day of measurement)/area at day 0] ×100 21, 22. Subsequently, tissues were dehydrated and processed for paraffin and with the means of microtome, 5 µm thick incisions were made and then stained by Toluidine Blue, and Hematoxylin and Eosin (H&E) 21, 23.For the histological study, the rats were euthanized and then full-thickness remaining wounds with the surrounding healthy skin were sampled and fixed in tubes containing formalin buffer 10% The evaluation of re-epithelialization, neovascularization, granulation tissue, inflammation and collagen alignment of the samples were accomplished by a pathologist who was blinded in grouping by evaluating H&E stained tissues and the given scores were based on Data analysisP<0.05 and analysis was performed by SPSS version 23 .The normal distribution of the variables was evaluated using the one-sample Kolmogorov-Smirnov test. Because the data were not distributed normally, the medians with interquartile range (IQR) were used for presenting the data. Kruskal-Wallis test followed by Dunn’s test was used to compare variables between the groups. Statistical significance was defined as EthicsThe experiment was approved by the Ethical Committee of Shahrekord University of Medical Sciences . We followed the ethics principles of working on animals.Measurement of phenolic and flavonoid compoundsThe phenolic compounds were totally 226.45 mg/g in Gallic acid equivalent.The flavonoid compounds were totally 9.95 mg Rutin equivalent/g.Wound closure rateMedian with Interquartile ranges (IQR) of wound closure rates in groups are shown in Histological findingsMedian with Interquartile ranges (IQR) of histological findings in groups are shown in Histological differences among groups with H&E staining, on day 15, are shown in Mast cell in the wound with Toluidine Blue staining is shown in This study aimed to evaluate the effect of white tea extract on the wound healing process in male Wistar rats  showed that despite flavonoids and antioxidant properties of white tea, it did not improve wound healing. 8, 24, 25. Some factors have effects on the wound healing process; such as age, sex, stress, alcohol, smoking, nutritional condition, and diabetes 4.After occurrence of a wound (which is an interruption of the continuity of cells of the skin), the wound healing process begins; in which the four overlapping phases including hemostasis, inflammation, cellular proliferation, and remodeling should be done in proper sequence and time frame 26. Various plants have been studied to evaluate their effects on the wound healing process. A blend of Alchemilla vulgaris and Mimosa had positive effects on re-epithelialization, collagen synthesis and angiogenesis 27. Ethyl acetate extract of Gmelina arborea has anti-inflammatory effects and could promote keratinocytes’ wound healing 28. Green tea extract could help wound healing of the surgical wound of rats 21. Iris forentina caused higher wound closure and density of fibroblast and collagen bundles in rats 22. The positive effects of these plants have been attributed to their phenolic compounds, especially the flavonoid components.Plant-based medicines are used by 80% of world’s population 29-31. Besides, polyphenols and white tea have a protective effect against wrinkles, sunburn and UV damages on the skin17, 19.Different studies showed the burn wound healing activity of polyphenols and antioxidants Therefore, the lack of positive effects in the present study is somewhat unexpected.henna, pomegranate, myrrh, the mixture of these three and Gentamycin on wound healing was compared. The most wound contracture and the least epithelialization duration were reported in the group treated with the mixture of plants 24. In another study, the effect of 10. Several properties have been attributed to flavonoids, such as anti-inflammatory properties 19. Inflammation is part of normal wound healing. Neutrophils, macrophages and T lymphocytes are the main cells of this phase in which neutrophils arrive at the wound location in 24-36 h, while, macrophages and T lymphocytes in 72 hours 32.Some other plants used for cutaneous wound treatment have flavonoid compounds P<0.05) less than Eucerin-treated, white tea 5% ointment (Eucerin)-treated and white tea 5% gel-treated groups; while, we do not expect inflammation until this day 32. Impaired wound healing has some complication such as displeasing scar formation and infection 21.Results of inflammation among the groups of this study  showed t6, 25, 32-38There are a lot of factors that can increase the risk of impaired wound healing and chronic wounds including genetic disorders like down-syndrome and ataxia-telangiectasia, aging, psychological stress by harming the immune system, malnutrition including zinc, vitamin B12, vitamin C and vitamin D deficiencies, the excess activity of macrophages through the release of pro-inflammatory cytokines, infection, diabetes through various means such as hyperglycemia, hypoxia and impaired immunity, coagulation defects, inadequate angiogenesis in patients with DM, and venous stasis and elderly 25.Other influential factors are foreign bodies, some diseases such as jaundice and uremia, obesity, and medications like glucocorticoids White tea 5% extract did not improve the wound healing process except neovascularization, attributed to the observed inflammation. The only factor ameliorated by white tea extract was neovascularization which may be due to polyphenols and antioxidant effects of white tea. There might be additional influential factors for impaired wound healing; therefore, additional studies are needed.


The conventional treatment of canine inflammatory bowel disease (IBD) includes corticosteroids, but they cannot contain the disease effectively in a percentage of patients. Still, their suppression can lead to a worsening. Moreover, the application of mesenchymal stem cells (MSCs) as an alternative has yielded promising results. However, they have been always infused after a washout period of any other immunosuppressants. Therefore, the feasibility and effects of the combination of stem cells and prednisone in IBD-dogs will be evaluated for the first time in this study. A single infusion of MSCs were administered to a group of IBD-dogs without any treatment and to another having prednisone treatment with poor response. The changes in two clinical indices, albumin and cobalamin concentration were assessed after one, three, six and 12 months. In both groups, an alleviation of the disease severity and an increase in albumin and cobalamin concentrations were observed at each visit. In parallel, the steroid dosage was gradually reduced until it was suppressed in all patients a year after the stem cell infusion. Therefore, the benefits of stem cell transplantation in dogs with inflammatory bowel disease receiving or not prednisone are significant and lasting.Mesenchymal stem cells have proven to be a promising alternative to conventional steroids to treat canine inflammatory bowel disease (IBD). However, their administration requires a washout period of immunosuppressive drugs that can lead to an exacerbation of the symptoms. Therefore, the feasibility and effects of the combined application of stem cells and prednisone in IBD-dogs without adequate response to corticosteroids was evaluated for the first time in this study over a long- term follow up. Two groups of dogs with IBD, one without treatment and another with prednisone treatment, received a single infusion of stem cells. The clinical indices, albumin and cobalamin were determined prior to the infusion and after one, three, six and 12 months. In both groups, all parameters significantly improved at each time point. In parallel, the steroid dosage was gradually reduced until it was suppressed in all patients a year after the cell therapy. Therefore, cell therapy can significantly and safely improve the disease condition in dogs with IBD receiving or not receiving prednisone. Furthermore, the steroid dosage can be significantly reduced or cancelled after the stem cell infusion. Their beneficial effects are stable over time and are long lasting. Inflammatory bowel disease (IBD) is the collective term for a group of chronic enteropathies characterized by persistent or recurrent gastrointestinal (GI) signs and inflammation of the GI tract . Even thTo treat this condition, immunosuppressants are commonly administered to patients that fail to respond to either dietary changes or antibiotics. The most widely used drug is prednisone, although studies have also been conducted with other immunosuppressive drugs, including cyclosporine, azathioprine and budesonide . HoweverThe application of MSC to treat immune-related gastrointestinal disorders have been investigated both in human ,6 and inClinical trials in small animals have mostly been designed to establish the efficacy and safety of the new therapy, so, a washout period before the treatment and the absence of any other medical treatment during the trial was required. On the contrary, in most human clinical trials conducted on Crohn’s patients, once efficacy and safety issues were solved, concomitant immunosuppressive agents were allowed, with their dosage being modified after the cell administration ,11,12. TTo be precise, the goal of this study is to establish if the co-administration of adipose derived mesenchymal stem cells (Ad-MSC) and prednisone would allow the prednisone dose to be subsequently reduced. Furthermore, we investigate whether the effects of combined Ad-MSC and corticosteroids are greater or lesser than the administration of Ad-MSC alone. Additionally, despite good results regarding the use of MSC in dogs and cats with IBD, their long-term effectiveness has not yet been evaluated and the number of cases in both studies has been very small. Due to this, the secondary aim of this work is to expand the number of dogs with IBD treated with stem cells and their clinical and laboratory follow-up in the medium and long term.Dogs diagnosed with IBD and treated in the Internal Medicine consultation of the Veterinary Teaching Hospital of the University of Extremadura (VTH-UEx) were included in the study, which was approved by the UEx Animal Care and Use Committee and by the Government of Extremadura . Prior information was provided, and all owners signed the written consent.−MSC group: Animals without any treatment at least 21 days before MSC administration.−P-MSC group: Animals treated with prednisone at the time of MSC administration . The starting dose of prednisone in dogs in this group was between 0.75 and 2 mg/kg per day. In addition, the prednisone dosage was assessed and reduced at the different time points post-treatment if CIBDAI and CCECAI scores dropped more than 30% of the previous value. If not, the dosage was maintained.The animals in this study were divided in two groups according to the received treatment:All owners were advised to feed their dogs with a hypoallergenic or novel protein diet to minimize diet interaction and variability.®, Spinreact, Barcelona, Spain), blood chemistry was performed on the Saturno 100 VetCrony®  automatic analyzer and, finally, the folate, cobalamin and TLI values were analyzed in the external laboratory Laboklin .The dogs in this study, prior to inclusion, had a history of chronic gastrointestinal signs (at least three weeks) and failed to respond to symptomatic therapies. IBD diagnosis was confirmed on the basis of routine diagnostic tests performed at the VTH-UEx. Referred animals were re-evaluated and those initial tests were repeated at the hospital. The diagnostic procedure included a detailed anamnesis, a complete physical examination and a complete blood count and biochemistry, including the measurement of albumin, folate, cobalamin and trypsin-like immunoreactivity (TLI). Blood hematology was carried out using an automatic analyzer , and the abdominal ultrasounds using the Philips® HD 11XEDS Ultrasound System . A urinalysis and stool analysis were performed on three consecutive days together with a Giardia test. Once extra-intestinal pathologies and parasitic and infectious diseases that could cause chronic digestive signs were ruled out, a digestive endoscopy (gastroduodenoscopy and ileocolonoscopy) was performed. Finally, mucosal samples endoscopically obtained were histopathologically analyzed, thus confirming the presence of inflammation. Food and antibiotic responsive enteropathies were excluded in all dogs according to the recommendations of the World Small Animal Veterinary Association Gastrointestinal Standardization.Subsequently, abdominal radiographs and a full abdominal ultrasound were performed. The radiographs were performed using the X-ray equipment Siemens Axiom MultiX MTThe criteria for patient inclusion were adult dogs (≥1 year of age) with histologically confirmed IBD and without adequate response to immunosuppressants.To establish the severity of each dog illness, the Clinical Inflammatory Bowel Disease Activity Index (CIBDAI)  and the Isolation, cell culture and characterization of adipose-derived mesenchymal stem cells were performed by ICTS Nanbiosis (Unit 14 at CCMIJU).n = 4) during conventional ovariectomy. The cells were washed twice with phosphate buffer saline (PBS) and digested at 37 °C for 30 min with 1.5% of collagenase type V  at 37 °C with agitation. The digested samples were washed twice with Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and filtered through a 40 µm nylon mesh. Cells were seeded onto tissue culture flasks and expanded at 37 °C and 5% CO2. Following 48 h in culture, the non-adherent cells were removed. Adhered cells were passaged at 80–90% confluence by trypsinization (0.25% trypsin solution) and seeded to a new culture at a density of 5000–6000 cells/cm2. Culture medium was changed every 4–7 days.Ad-MSC were obtained from subcutaneous adipose tissue from the falciform ligament of healthy donors  as previously described [Cells were expanded over three passages and then cryopreserved in fetal calf serum (FCS) with 10% of dimethyl sulfoxide (DMSO). The MSC phenotype of the adherent cells was verified according to the International Society for Cellular Therapy guidelines . Flow cyescribed .A second expansion was performed to obtain the final product for in vivo administration. The day of the treatment, cells were carefully thawed at 37 °C, FCS and DMSO were removed by centrifugation of the cells, and the pellet was resuspended in 50 mL of vehicle . Additionally, a mycoplasma test was performed in all cell lines.6 cells/kg body weight. For the administration of Ad-MSC, thawed cells were resuspended and diluted in physiological saline (SSF) to a final volume of 100 to 250 mL . The infusion was administered through a peripheral IV cannula over an average of 30 min. All animals were monitored before, during and 1 h after administration.Patients received an intravenous single dose of 4 × 10Just prior to the administration of the Ad-MSC (T0), clinical exam, hematology, serum biochemistry and abdominal ultrasound were performed, and CIBDAI and CCECAI scores, cobalamin  and albumin  serum concentrations, and prednisone dosage for the P-MSC group were obtained.Patients were followed up at 30 (T1), 90 (T3), 180 (T6) and 365 (T12) days after cell infusion, and the same parameters were assessed at each of the time points and compared with the previous values.Clinical remission was defined as a decrease of over 75% in the CIBDAI and CCECAI T12 scores. Indeed, animals showing CIBDAI or CCECAI scores lower than 3 are considered to be in remission of the disease in this study.Statistical analysis was performed using the Sigma Plot 12.0 Extract Graphs and Data Analysis software (USA/Canada). First, a Shapiro-Wilk test was performed to analyze the normality of the data distribution. All variables analyzed  had a non-Gaussian distribution. Data are presented as mean values ± standard deviation.For each group, differences in data before and after treatment at each control  were compared using a Tukey test.Finally, to determine the differences between values from both groups for each studied parameter at each visit, a Mann-Whitney U test was carried out.p < 0.05.Statistical significance was set at p = 0.056) or weight (p = 0.673) between both groups , followed by German Shepherd (12.5%), Yorkshire Terrier (12.5%), French Bulldog (9.4%) and Boxer (6.3%). The rest of the breeds, representing 3.12% each, were Beagle, West Highland White Terrier, American Staffordshire Terrier, Bichon Maltese, Pitbull Terrier, Poodle, Siberian Husky, Spanish Mastiff, Golden Retriever, Greyhound, Pomeranian, Cocker Spaniel and Spanish Water Dog. There were no statistically significant differences in age . Clinical signs showed, from highest to lowest frequency, were watery diarrhea, pasty stools, decreased appetite/anorexia, weight loss, vomiting, increased frequency of defecation, apathy, hematochezia, melena, ascites and hematemesis. During the initial evaluation (T0), each patient showed 4 symptoms on average. After Ad-MSC administration, the number of symptoms decreased in most patients from both groups, presenting an average of 2 symptoms at T1, 1 at T3 and T6 and no symptoms at T12.Adipose-derived mesenchymal stem cells were isolated and in vitro expanded as previously described . The celAccording to the clinical indices, the study group at T0 included 1 animal with medium, 12 with moderate, 10 with severe and 9 with very severe IBD. Baseline CIBDAI and CCECAI population mean scores were 8.75 ± 3.37 (moderate IBD) and 9.80 ± 3.42 (severe IBD), respectively.p < 0.05). In the prednisone and cells joint treatment group, that reduction was only significant for the values obtained at T3, T6 and T12 when compared to T0 (p < 0.05)  .p < 0.05) (CCECAI scores fell 8.06 points in the MSC group and 9.91 in the P-MSC group a year after . In both < 0.05) .There were no significant differences among the values obtained from the post-cell treatment follow-up controls in any index or group .Indices reduction from baseline were significantly greater in the MSC group at T1 and T3 than in the combined therapy group . All aniClinical remission was achieved by 57% of the animals (11/19) in the MSC group and by 30% (4/13) in the P-MSC group at T1. At T3, 68% (13/19) of the MSC-patients and 61% (8/13) of the P-MSC patients achieved clinical remission. Six months after the administration of the MSC (T6), remission was observed in 84% (16/19) of the animals in the MSC group and also in 84% (11/13) of the animals in the P-MSC group. Finally, in the annual check-up (T12), 100% of the animals achieved remission in both groups.At the time of diagnosis, 13 of the 32 patients had hypoalbuminemia (40.6%), 5 of them in the MSC group and 8 in the P-MSC group. The baseline mean albumin value of the study population was 2.67 ± 0.75 g/dL. The T0 albumin concentration was significantly lower in P-MSC animals than in MSC animals .After cell administration, the albumin concentration improved gradually in both groups, with only one dog being hypoalbuminemic at T1 and none at the following visits in the MSC group. In the prednisone group, the five animals remained hypoalbuminemic at T1, but decreased to 3 at T3, 2 at T6 and none at T12.A year later, the albumin concentration had increased by 0.57 g/dL in the MSC group and 0.81 g/dL in the P-MSC group . The incPrior to cell treatment, 19 of the 32 patients in the study (59%) had hypocobalaminemia, 11 in the MSC group and 8 in the P-MSC group. Mean cobalamin concentration at T0 for the study group was 292.34 ± 189.38 pg/mL. Even though mean values of cobalamin in the MSC group were lower than in the prednisone group at T0, the difference was not significant .After a month, 7 patients from the MSC group and 5 from the P-MSC group remained hypocobalaminemic. Three months later, they decreased to 3 and 2 in the MSC and P-MSC group, respectively. At T6 and T12, the cobalamin concentration was normal in all the dogs of the cell group. In the prednisone group, hypocobalaminemia persisted in 2 dogs at T6 and in 1 dog at T12.A year after MSC infusion, cobalamin had increased by an average of 316.38 pg/mL in the MSC group and only by 97.92 pg/mL, on average, in the P-MSC group . In the After the administration of MSC, the prednisone dose was progressively reduced over the successive controls . CompareMSC have been used in veterinary medicine in immune-mediated diseases due to the important role they play in regulating the immune system. Studies have been conducted using this cell therapy in patients with canine atopic dermatitis, keratoconjunctivitis sicca, granulomatous meningoencephalitis, feline chronic gingivostomatitis, feline asthma and canine and feline chronic IBD ,17.Previous preliminary studies on feline and canine IBD offered promising results, reporting a significant improvement in clinical signs ,9. Thus,Canine IBD is routinely treated with steroids, cyclosporine or azathioprine, separately or combined . In someIn human medicine, numerous studies in which MSC of different origins were administered to IBD patients in combination with immunosuppressant therapy have been carried out, concluding that their co-administration was safe, the steroid dosage significantly decreased and the patients’ conditions also improved significantly ,11,12. LDifferent investigations have shown that the co-administration of MSC and other IBD treatments leads to synergy, can repair the mucosa and can improve intestinal inflammation . SchneidOn the other hand, this study also shows that both cell therapy modalities  seem to be equally effective with some minor differences. Even though the clinical indices decreased considerably after the MSC administration, their fall was higher in the combined therapy group, even from a worse starting situation . As someHypoalbuminemia affected a higher number of dogs in the combined therapy group. In fact, it was the only group that started off from an average albumin concentration below the normal level. This condition has been described more frequently in patients with advanced disease who require the administration of immunosuppressants . FurtherAccording to various studies, the prevalence of hypocobalaminemia in IBD patients varies between 19% and 38% . In thisMSC have been reported to secrete a variety of immunosuppressive molecules, thereby reducing inflammation in a generalized manner. They also promote wound healing and tissue regeneration by secreting the so-called transforming growth factor β (TGF-β) and fibroblast growth factor. Furthermore, these cells can differentiate into fibroblasts and endothelial cells to form granulation tissues. Therefore, they have regenerative and anti-inflammatory qualities, which are beneficial for IBD .However, the short duration of earlier studies  ,9 left tThe main limitation of the study is the small number of recruited patients, since most dogs with chronic enteropathies responded to special diets or antibiotics. Another weakness might be the lack of a prednisone-treated control group. However, this study is not intended to assess the efficacy of MSC when compared to steroids, but to prove the feasibility of the combination of both and to restate the utility of cell therapy for canine IBD by increasing the number of MSC-treated dogs and the follow-up period.The origin and type  of MSC, their laboratory preparation and the method of administration  can also affect the final outcome of the treatment . TherefoAd-MSC therapy can significantly and safely improve the disease condition in dogs with IBD both without treatment and receiving a stable steroid dose. Furthermore, the steroid dosage can be significantly reduced or cancelled after Ad-MSC infusion. Their beneficial effects are stable over time and are long lasting.


FST and hapFLK, respectively) between populations, differences in long stretches of consecutive homozygous sequences (ROH), and differences in allele frequencies within populations (composite likelihood ratio (CLR)) to identify inter- and intra-populations traces of selection in two Iranian indigenous chicken ecotypes, the Lari fighting chicken and the Khazak or creeper (short-leg) chicken.Various regions of the chicken genome have been under natural and artificial selection for thousands of years. The substantial diversity that exits among chickens from different geographic regions provides an excellent opportunity to investigate the genomic regions under selection which, in turn, will increase our knowledge about the mechanisms that underlie chicken diversity and adaptation. Several statistics have been developed to detect genomic regions that are under selection. In this study, we applied approaches based on differences in allele or haplotype frequencies , reproduction (ESR1 and CALCR), disease resistance , behavior pattern , and morphological traits (IHH and NHEJ1).Using whole-genome resequencing data of 32 individuals from the two chicken ecotypes, approximately 11.9 million single nucleotide polymorphisms (SNPs) were detected and used in genomic analyses after quality processing. Examination of the distribution of ROH in the two populations indicated short to long ROH, ranging from 0.3 to 5.4 Mb. We found 90 genes that were detected by at least two of the four applied methods. Gene annotation of the detected putative regions under selection revealed candidate genes associated with growth (Our findings show that these two phenotypically different indigenous chicken populations have been under selection for reproduction, immune, behavioral, and morphology traits. The results illustrate that selection can play an important role in shaping signatures of differentiation across the genomic landscape of two chicken populations.The online version contains supplementary material available at 10.1186/s12711-021-00664-9. Gallus gallus) in the south and Southeast Asia [FST) [Chickens are raised for different purposes, including meat and egg production, as well as for entertainment, in diverse geographical areas. Several studies have reported the multiple times when and multiple places where the domestication of chicken from the Jungle fowl species occurred. However, most of these studies suggested that domestic chicken originated from the Red Jungle fowl  , cross-pia [FST)  and hapFia [FST) . Since eia [FST) , 17.FST, and have led to the identification of a range of candidates genes that contribute to adaptation to hot climates, reproduction [FST method does not account for the hierarchical structure of populations and assumes that the subpopulations have derived independently from the same ancestral population [To decipher the genetic mechanisms that are involved in domestication and in the phenotypic differentiation of individuals belonging to the same species, detection of signatures of selection using different statistics has been performed in various species, including cattle , 19, sheoduction , immune oduction , fat depoduction . Howeverpulation . To addrpulation , was intpulation . The Happulation , 30.FST mapping in African chicken breeds and indigenous ecotypes detected signatures of selection related to adaptation to heat, immunity, calcium ion binding, behavior, etc. [ROH are contiguous lengths of homozygous segments of the genome where the two identical haplotypes have likely been inherited from a common ancestor. Fleming et al.  employedor, etc. .Indigenous chickens are notable for their capacity to adapt to their respective climates and for their disease resistance and it is essential to preserve them as valuable genetic resources. Since Iranian indigenous chickens have never been subjected to selection programs for production traits, mapping the genes and quantitative trait loci (QTL) that are associated with such traits would be useful for future breeding programs. However, there is limited knowledge about the genomic regions and genes involved in economic traits of interest and in adaptation to harsh environment conditions .FST, ROH, hapFLK, and CLR.There is a worldwide diversity of domesticated chickens that differ in morphology, physiology, and behavior . Lari anThirty-two chickens were sampled from the south and southeastern regions of Iran (15 Lari and 17 Khazak). Each ecotype was sampled in multiple village flocks in order to minimize family structure or high levels of co-ancestry between individuals. Extraction of genomic DNA from blood collected in EDTA-coated tubes was carried out using the standard phenol–chloroform protocol . Quantithttp://broadinstitute.github.io/picard/). In order to improve the accuracy of downstream processing steps, local alignment of the sequences around InDels was performed by RealignerTargetCreator and IndelRealigner in the GATK software [Paired-end reads were aligned to the chicken reference genome  using the mem algorithm in the Burrows-Wheeler Aligner (BWA) software , the ressoftware . To adjusoftware  was usedTo investigate the genetic structure and differentiation of the target populations, a phylogeny tree was created by the maximum likelihood method using the PHYLIP package in SNPhylo . A princThe CLR test detects differences in allele frequencies along a chromosome between a neutral and a selective sweep model, which is able to detect variants that are close to fixation . We perfPutative genomic regions under selection within each population were detected by the “Runs of homozygosity” function of the Plink software. The parameters were set according to Ceballos et al. , such thThe frequencies and length of ROH across the genome can differ between individuals. For instance, a longer ROH implies recent inbreeding, while a shorter ROH indicates more ancient common ancestors in the pedigree , 49. We FST statistic, which is a method based on the differentiation in allele frequencies between populations, was used to identify genomic footprints of selection between the two populations. This approach has been applied in numerous studies to investigate the underlying genomic mechanism of variation between populations [FST statistic between the two populations, we used the VCFtools software [FST estimator [FST values were estimated in 50-kb sliding windows along each chromosome, with a step size of 25 kb. The degree of genetic divergence and differentiation between populations (FST) ranges from 0 to 1, where values close to 0 refer to a low genetic differentiation, i.e. the two populations have no genetic differentiation and share many fixed loci and values close to 1 refer to a stabilized difference between populations [FST values were set equal to zero because negative values do not have a biological interpretation [FST (mFST) values as divergent selection footprints.We compared two indigenous chicken populations that were phenotypically different. The ulations , 50. To software , based ostimator . Pairwisulations . In thisretation . We conshttps://forge-dga.jouy.inra.fr/projects/hapflk. We used 10 clusters (-K 10) for the fastPHASE cross-validation procedure to obtain haplotype diversity [The hapFLK statistic which is a haplotype-based approach, was calculated according to the methodology described by Fariello et al.  using thiversity . The hapSince hapFLK values for each SNP along the genome approximately follow a normal distribution, to robustly evaluate the normal distribution parameters, hapFLK values were standardized to evaluate the p-value for each SNP using the formula: package . SNPs thTo control and avoid the detection of likely false positives by different methods, we focused on signatures of selection that were identified by at least two of the four approaches and on those that were found in peak points in the hapFLK analysis as putative signatures of selection. The genes located in these regions were determined by the Variant Effect Predictor tool (release 101)  and funchttp://www.animalgenome.org/cgi-bin/QTLdb/GG/index). Then, we used the regioneR [In order to examine the overlap of the putative genomic regions under selection with previously known QTL, we downloaded the QTL data from the chicken QTL database  of the short reads were properly mapped to the chicken reference genome . The average sequencing depth for the 32 individuals was 7.1 (6.9 for Khazak and 7.3 for Lari). In total, 17,287,526 SNPs were detected from the whole-genome data in the initial SNP calling. After quality control, 11,873,011 SNPs across all individuals were retained. Of those, about 6.5 million SNPs were shared between the two ecotypes, and 2.9 and 2.8 million SNPs were specific to the Lari and Khazak ecotypes, respectively.Using the 11,873,011 SNPs, a maximum likelihood phylogenic tree was constructed and the evolutionary divergence of populations was visualized. Although there were sub-clusters in each population, the two populations segregated into two distinct clusters  than the Khazak ecotype  gene , biosynthesis of fatty acid and abdominal fat deposition (ELOVL2 and MAOA), and immune traits , while those identified in the Lari chickens are involved in regulation of energy homeostasis (AGRP), immune (APBB1IP), muscle development (HDAC9), wound healing (MMP13), metabolic regulation and reproduction (TSHR), and behavioral traits (AGMO and PSEN1). In the CLR test, enriched GO terms were revealed for the biological process, cellular components, and molecular functions categories. Functional terms included those involved in epithelial cell proliferation, Golgi organization, and cell differentiation  were identified (110 in the Lari and 65 in the Khazak population)  gene. For the Khazak population, one consensus ROH was identified on chromosome 8  that was common to 16 individuals and included long non-coding RNA transcripts.Annotation analysis of consensus ROH revealed 282 protein coding genes , of which some contained the terms axonogenesis (GO: 0007409), visual perception (GO: 0007601), blood vessel maturation (GO: 0001955), and response to hypoxia (GO: 0001666)  (mFST = 0.541) and a protein-coding region on chromosome 2  that spans the AGMO gene (FST = 0.538) , ALPK1 , S1PR1 (sphingosine-1-phosphate receptor 1), and ITK (IL2-inducible T-cell kinase), which mapped to chromosomes 1, 4, 8, and 13, respectively. Furthermore, our results indicate that several genes in the detected regions are associated with growth and reproductive traits, such as DCN (decorin), MEOX2 (mesenchyme homeobox 2), HDAC9 (histone deacetylase 9), SGCZ , LARGE (like-glycosyltransferase), and CITED4  , 27 (6.48–7.69 Mb), and 16 (2.40–2.75 Mb).Results of the hapFLK test are shown in Fig. BF1, TAP1, DMB2, BLB1, BLB2, TAPBP, BRD2, BLEC1, C4, IKZF3, DMB1, and IL4I1) and in morphological traits such as the creeper (IHH and NHEJ1) and comb traits (MNR2). In addition, important genes relevant to growth and reproductive traits  were detected in several other significant regions  was smaller than the genomic ROH coverage reported for a commercial broiler line (130.9 Mb on average) , which c Mb was sormation , 67–69. ormation , 70. Theormation .FST and hapflk (inter-population), and ROH and CLR (intra-population), to identify signatures of selection for two phenotypically different chicken ecotypes. Depending on the nature of the information, each of these statistics has its advantages and disadvantages and may capture a specific pattern of selection [FST statistic has been widely applied in various studies, is more powerful for the detection of complex events, but it does not take the hierarchical structure of subpopulations into account; this limitation has been fixed in the hapFLK statistic, which is robust with regard to evolutionary processes, such as bottlenecks and migration, and has the ability to uncover a hard selective sweep, i.e. a new beneficial mutation that rises in frequency and spreads quickly to complete fixation. The CLR statistic can detect selective sweeps that are close to fixation, whereas the short ROH (~ 1 Mb) that are detected by consensus ROH (cROH) analysis mostly arose through selective pressure on identical-by-descent genomic regions from distant ancestors [Identifying positive signatures of selection can provide valuable information about the influence of selection on adaptive, productive, and morphological characteristics. In this study, we applied four procedures, election , 17, 71.ncestors . Howeverncestors , 68. Thencestors .DCN (decorin) and MEOX2 (mesenchyme homeobox 2) were good candidates, as both were detected by three of the four applied methods. DCN encodes a connective tissue protein, i.e. a multifunctional proteoglycan that enhances skeletal muscle tissue proliferation and differentiation by interfering with myostatin activity [DCN is also involved in modulating collagen assembly and bone mineralization [DCN expression increases in response to exercise and muscle contraction, which contribute to muscle hypertrophy [DCN in a gender-dependent manner, resulting in greater impact in men than in women [MEOX2 is a homeobox gene and is associated with skeletal muscle tissue and bone development [MEOX2 reduces muscle mass and causes a developmental defect in the limb musculature [MEOX2 affects muscle fiber metabolism and muscle size in adult mice [The Lari fighting chickens are characterized by an amazing level of activity. Cockfighting is a form of exercise in which the chicken requires mighty muscle and skeletal structure to run and jump. Thus, we hypothesized that some candidate genes could be connected with muscle growth, hypertrophy, and limb development. Along that line, the two genes activity . DCN is lization . Previouertrophy , 76. Furin women . MEOX2 ielopment . In miceculature . It has ult mice .CACNB1  gene on chromosome 27, with a P-value lower than 2.31 × 10–11. CACNB1 is known to be associated with body weight in broilers [In meat-type chickens, breast muscle is the most valuable carcass component and is one of the most important economic traits. The Lari chickens, with their wide breast and heavy weight (3–4 kg), can be considered as a meat-type chicken. In the haplotype-based analysis (hapFLK), we applied a stringent significant threshold to control false positive signals. One of the most striking signatures of selection was detected in a region that contained the broilers  and to pbroilers .ESR1 gene on chromosome 3 as a putative signature of selection for reproductive traits in the Khazak ecotype. In birds, the theca cells are responsible for the synthesis of estrogen in the ovarian follicles. Estrogen regulates the synthesis of egg white and yolk proteins, calcium mobilization, and reproductive behavior, and is necessary for folliculogenesis. The effect of estradiol on folliculogenesis is mediated by the estrogen receptors α and β, which are encoded by the ESR1 and ESR2 genes, respectively [ESR1 gene is higher than that of the ESR2 gene. ESR1 is reported to be associated with ovarian functions, especially during follicular development [Compared with meat-type chickens, the low weight of the Khazak chickens could be an asset because their energy consumption for maintenance will be lower and, thus, most of their feed intake will be dedicated to egg production. Gene annotation of the putative regions identified in the cROH and CLR analyses identified the ectively . In the elopment , 84, andelopment , 86. Thielopment , 88.FST and CLR analyses for the Khazak ecotype revealed another gene that is involved in reproduction traits, CALCR . The calcitonin receptor binds to calcitonin, a polypeptide hormone, and is associated with regulation of follicular maturation in the chicken ovary [Both the en ovary . In addien ovary , 91.In the putative regions of signatures of selection, several QTL were previously detected for economically important traits, such as growth, abdominal fat weight, drumstick and thigh muscle weight, egg number, and egg production rate. Since no breeding program has ever been implemented to improve production traits in the Iranian indigenous chickens, the identification of genes and QTL that are associated with these traits in these populations may be important for designing breeding plans in the future.FST windows. For example, the S1PR1 (sphingosine-1-phosphate receptor 1) gene was identified on chromosome 8 and is known to encode a lipid regulator that is involved in processes such as immune response [S1PR1 can reduce morbidity and mortality in H5N6-infected chickens by suppressing the induction of cytokines, chemokines, and pattern recognition receptors (PRR) [S1PR1 gene plays an essential role in inflammatory responses to infection with the Newcastle disease virus [ALPK1 , which is a member of the group of atypical kinase genes [Chickens raised in village regions are under different environmental conditions that can affect their productive performance. Natural selection pressure has led to genetic adaptations in village chickens that allow them to survive in the harsh and diverse environmental conditions . It has response  and to cresponse . Activatrs (PRR) . It has se virus , 96. Anose genes . It contse genes , 99.B (MHC-B). The MHC is a group of polymorphic genes with a central role in the immune system, particularly in resistance to infectious diseases [BF1, BF2, TAP1, DMB2, DMA, BLB1, BLB2, TAPBP, BRD2, BLEC1, C4, IKZF3, BZFP1, CENPA, CYP21A1, LTB4R, HEP21, TRIM7.2, ZNF692, TRIM7.1, TRIM27.1, TRIM27.2, TRIM39.2, TRIM41, TRIM39.1, and GNB2L1 genes. MHC-B haplotypes are important for resistance or susceptibility to infectious diseases such as Marek's disease, Russian sarcoma, and avian leukemia [In the hapFLK analysis, the strong selective sweep that was detected on chromosome 16 encompasses important genes related to immune traits. Miller and Taylor Jr  reporteddiseases –104. In leukemia , 106.FST and hapFLK) revealed potential candidate genes that are relevant to immune traits. This was expected because the Lari and Khazak ecotypes live in different eco-climates. Moreover, because the Lari chickens are used for entertainment and fighting purposes, they have a higher economic value than the Khazak chickens and thus, they probably receive more healthcare from their owners.The population differentiation analyses (IHH (Indian hedgehog) and NHEJ1 (non-homologous end-joining factor 1), and has been documented to be the causal genes of the creeper trait in chickens [IHH and NHEJ1 genes on chromosome 7 has a significant role in the Cp phenotype of Japanese chickens.The Khazak chickens are characterized by a small body size and, compared to the Lari chickens, the length of their leg is much shorter (4 cm vs. 16 cm), such that in the local language, they are known as a creeper chicken. Within the identified putative regions of signatures of selection, two genes that are known to influence the creeper trait were detected in the whole-genome scan using the haplotype differentiation analysis. The strongest signal was observed on chromosome 7 between 22.29 and 22.32 Mb, which included 79 SNPs. This region spans two important genes, chickens , 108. Thchickens . Recentlchickens  demonstrFST, CLR, and ROH) identified the same signature of selection on chromosome 2, which has the highest mFST value (0.541) and a significant CLR value (8.80). This consensus ROH region was present in 73.3% of the Lari population analyzed. This region includes the AGMO  gene, which encodes the only enzyme that cleaves the O-alkyl bond of ether lipids and plays a role in the disabilities and neurodevelopmental disorders in humans [AGMO gene that are associated with behavioral patterns in the Chinese gamecock chickens. We also detected the GNAO1 gene in a cROH region on chromosome 11 that was present in 47% of the Lari chickens. It has been shown that the GNAO1 gene is involved in the aggressive behavior of the Xishuangbanna fighting chicken and is under selection in that population [PSEN1 (presenilin 1), which is located on chromosome 5 and was detected in the FST and CLR analyses of the Khazak ecotype. The PSEN1 gene has a role in neuropsychiatric diseases in humans [Aggressive behavior has been evolutionarily conserved during chicken domestication  and is pn humans , 112. Lun humans  highlighpulation , 115. Ann humans  and may n humans .FST windows overlapped with the detected cROH across the genome. In our study, we detected several putative regions of signatures of selection and genes that were shared by at least two of the four methods used, but we did not observe overlapping regions between the FST and hapFLK approaches because these two methods recognize specific different patterns of signatures of selection. The detection of shared regions under selection by different methods could provide persuasive evidence about the effect of selection on a specific region in the genome, which may be associated with critical functional traits. In addition, the putative regions of signatures of selection that overlap with known QTL can enhance the accuracy of detected signatures of selection [Since each statistical approach is able to detect specific signatures of selection across the genome, it is possible that they do not detect the same region for a given signature of selection . Howeverelection . HoweverWe implemented four intra and inter-population methods to detect signatures of selection using whole-genome resequencing data in two indigenous chicken ecotypes. Our results revealed several putative footprints of selection that harbor candidate genes associated with disease resistance, growth, reproductive, morphological, and behavioral traits. Although indigenous chicken ecotypes are not always considered for large-scale commercial purposes, they have good potential to survive in harsh environmental conditions. Our findings enhance our understanding of the relationships between phenotype differentiation and genotypes among different breeds.Additional file 1: Table S1. Summary of the distribution of ROH lengths along the genome per animal. An excel file containing the number of ROH, total size of genome covered by ROH (kb) and average of ROH length (kb) per animal.Additional file 2: Table S2. List of candidate genes that overlap with regions identified by the CLR method in the Lari and Khazak ecotypes.Additional file 3: Table S3. List of the genes that were identified simultaneously by at least two of the four applied methods. This table represents the identified common genes shared by at least two of the four methods used, FST, hapFLK, CLR and ROH, (L: Lari ecotype and K: Khazak ecotype).Additional file 4: Table S4. Summary of GO terms related to biological processes resulting from the analyses of cROH regions, CLR, FST and hapFLK in the Lari and Khazak ecotypes. This table represents gene ontology (GO) terms that are significant , (L: Lari ecotype and K: Khazak ecotype).Additional file 5: Table S5. List of consensus ROH pools for each ecotype. This file contains information about family identification (FID), individual identification (IID), chromosome (CHR), start position (BP1) and end position (BP2), segment size (KB) and number of SNPs in each ROH.Additional file 6: Table S6. List of candidate genes that overlap with regions identified by the ROH method in the Lari and Khazak ecotypes.Additional file 7: Table S7. List of candidate genes that overlap with regions identified by the FST method.Additional file 8: Table S8. List of candidate genes that overlap with regions identified by the hapFLK method.Additional file 9: Table S9. Overlaps between the reported QTL in the QTL database and the detected candidate regions by FST, hapFLK, CLR and ROH analysis. This file presents the overlaps between the traits and the position of the known QTL regions in chicken with the position of the identified putative signatures of selection by the four methods.Additional file 10: Figure S1. Results of the overlap analysis between the regions of signatures of selection (set A) and known QTL regions in chicken (set B). The y-axis shows the number of overlaps between the two datasets. AinB refers to the region in A encompassed by a region in B; BinA refers to the region in B encompassed by a region in A; AleftB and ArightB refer to the end and the start of the region of signatures of selection that overlap with the beginning and the end of a region of QTL, respectively.