Although person-centered care (PCC) ensures high-quality care for patients, studies have shown that it is unevenly applied in clinical practice. The extent to which future health care providers are currently offered education in PCC at their universities is unclear. We aimed to clarify the PCC content offered to students as a basis for their understanding by exploring the PCC content of Swedish national study programs in medicine, nursing, occupational therapy, and physiotherapy.n = 48) with accreditation in medicine (n = 7), nursing (n = 25), occupational therapy (n = 8), or physiotherapy (n = 8) at a single time point. All national study programs (n = 4), local program syllabuses (n = 48), and local course syllabuses (n = 799) were reviewed using a 10-item protocol.Using a qualitative document analysis design, we sampled the steering documents from all higher education institutions  included PCC in their intended learning outcomes. Eight of the 799 local course syllabuses (1%) contained course titles that included the phrase ‘person-centered care,’ and another 101 listed 142 intended learning outcomes referring to PCC. A total of 21 terms connected to PCC were found, and the term ‘person-centered care’ was most commonly used in the nursing programs and least commonly in the medical programs.There is a broad range in how the national study programs in Sweden have incorporated PCC. The implementation has been driven by a bottom-up strategy. A deliberate and standardized strategy is needed to ensure full implementation of PCC into clinical curricula in higher education. Patients, caregivers, and the World Health Organization have all called for a shift towards person-centered care (PCC) , 2, moviSeveral frameworks exist for PCC –11, inclA successful implementation strategy for PCC requires that several actors from different health and social care sectors work together in a unified approach to mutually drive forward change , 19, 20.The overall aim was to explore the PCC content in four Swedish national study programs: medicine, nursing, occupational therapy, and physiotherapy.This study used a qualitative document analysis design .Swedish national study programs are funded publicly, and the education system is governed legally by the Swedish Higher Education Act  and HighIn spring 2020, the Swedish national study program (level I) in medicine comprised 330 HECs, which corresponds to 11 semesters of full-time studies provided at seven higher education institutions (HEIs). The national study programs in nursing, occupational therapy, and physiotherapy (level 1) comprise 180 HECs or six semesters of full-time studies. The national nursing program is offered at 25 HEIs and the national programs in occupational therapy and physiotherapy at 8 institutions each. In spring 2020, a total of 10,895 students were admitted to these four national study programs: 2155 to the program in medicine, 7218 to the program in nursing, 693 to the program in occupational therapy, and 829 to the program in physical therapy .To extract data from national steering documents, such as national study programs (level 1), local program syllabues (level II), and local course syllabuses (level III), a protocol was developed in three phases.A steering committee consisting of three researchers . In terms of the researchers’ prior understanding, all had previous research experience with PCC and were affiliated with University of Gothenburg Centre for Person-Centred Care (GPCC). Moreover, they all had previous teaching experience and knowledge and understanding of steering documents at the three levels. Their task was to develop a protocol to examine the steering documents.A crucial step in developing a protocol was to a priori agree upon the conceptual definitions of PCC to include in the study. PCC is described within several frameworks and, even though the authors adhered to the description of PCC by Ekman et al. , our resA syllabus governs and controls the content and learning outcomes of education programs; thus, it highlights the values of a specific society , 38. AccThus, the steering group wanted to examine the total number of intended learning outcomes referring to actors and linked to context, actions, and relationships in the national study plan (level I), a total number of included local program syllabuses (level II) and local course syllabuses (level III), intended learning outcomes, courses, and course titles. Furthermore, the total distribution of the intended learning outcomes by semester in the study programs and how they were distributed around what students should have achieved at the end of the course regarding knowledge/comprehension and skills judgment/attitude was calculated. Finally, there was an interest in examining the contents describing PCC and variants of this term.n = 48), and in the third level of the protocol five questions to examine the local course syllabuses  retrieved data on the national study programs and syllabuses at all three levels from the university and college websites between January and May 2020. Thereafter, the researchers separately examined the national study program (level I), local program syllabuses (level II), and local course syllabuses (level III) using the protocol described in Table According to previous authors , documenThe steering documents were read  several times to achieve overall familiarity and a picture of their manifest content .Meaning units  were selected by the researchers  using a deductive approach. PCC context was searched for in the the local program (level II) and local course syllabuses (level III) (questions 2 and 6), as well as intended learning outcomes , courses (question 4), course titles (question 5), semesters (question 8), headings of learning levels (question 9), and terms connected to actors, context, actions, and relationships (question 10). Identified meaning units were then extracted, condensed, and labeled with a code. Finally, the codes were sorted and abstracted into categories. Deductive coding was used to assign an appropriate heading for an intended learning outcome (question 9). The unassigned headings were coded into a code map based on Bloom’s taxonomy . UnassigFrequencies and percentages were calculated to describe the total number of local program and course syllabuses (questions 2 and 6), intended learning outcomes , courses (question 4), course titles (question 5), semesters (question 8), headings of learning levels (question 9), and terms connected to actors, context, actions, and relationships (question 10) .To ensure trustworthiness, several actions were planned a priori. Credibility was ensured by the fact that all researchers  who collected data have experience reading and interpreting steering documents in higher education (reflexivity). The data collection period lasted for 5 months (prolonged engagement). All researchers in the study participated in the discussion on how the results should be interpreted and understood (member checking). To account for the study's dependability, there was a description of how the steering documents were retrieved, identified, analyzed, and described (investigator triangulation). Confirmability was secured by describing how the study protocol was created and what phenomenon  we looked for in the steering documents (audit trail). Confirmability was ensured by describing what the study's phenomenon was, which control steering documents constituted data, what the study context was, and how data were analyzed (audit trail). Authenticity was ensured by all researchers using the study protocol (audit trail) and all research group members participating in the discussions of the results (member checking) , 44.n = 4), we found no content referring to actors and linked to context, actions, and relationships in the intended learning outcomes.In the national study programs (n = 48), 8 (1%) had course titles referring to actors and linked to context, actions, and relationships. All identified course titles were in the nursing program. For example, two HEIs in nursing used the titles, ‘Person-centered care for mental illness’ and ‘Person-centered nursing, caring approach, and communication.’Of the 48 approved local program syllabuses, 7 (15%) included nine local intended learning outcomes of content referring to actors and linked to context, actions, and relationships. For example, one local program syllabus in medicine had the local intended learning outcome, ‘To use a patient-centered approach in clinical work,’ one local study program in occupational therapy had the local intended learning outcome, ‘To apply person-centered and reflective approach,’ and one local study program in nursing used the local intended learning outcome, ‘Have the competence to implement PCC.’ Of the 799 local course syllabuses identified in the local program syllabuses  included 142 intended learning outcomes with content referring to actors and linked to context, actions, and relationships. For example, one of the intended learning outcomes in medical education was given as, ‘Using patient-centered conversation methodology, initiate the conversation and clarify the reason for the visit, including thought, concern, and desire.’ Another example, from the physiotherapist program, was, ‘Reflect on the importance of good communication and collaboration for effective person-centered care.’n = 7) had most intended learning outcomes with content referring to actors and linked to context, actions, and relationships in semesters 4, 5, and 6 (each 17%). Furthermore, this study program had 4% intended learning outcomes with content referring to actors and linked to context, action, and relationship in semesters 7, 8, 9, and 11. However, HEIs of nursing (n = 25) had most intended learning outcomes with content referring to actors and linked to context, action and relationship (23.5%) in the first semester. HEIs of occupational therapy (n = 8) had most of their intended learning outcomes with content referring to actors and linked to context, actions, and relationships (33.3%) in semester 4 and HEIs of physiotherapy (n = 8) in semesters 4 and 6 (each 30%). Overall, the HEIs (n = 48) had most of their intended learning outcomes with content referring to actors and linked to context, actions and relationship in the sixth semester (28.2%).Fourteen of the 48 HEIs 29%) did not have any content referring to actors and linked to context, actions, and relationships in their intended learning outcomes  and ‘person-centered care’ (23 times). The most common terms in the medical study program were ‘patient-centered consultation’ (4 times), whereas the physiotherapy study program used ‘person-centered approach’ (2 times), ‘person-centered care’ (2 times), and ‘person-centered goal’ (2 times). In the occupational therapy study program, the term ‘client-centered practice’ (5 times) was most commonly used  contained more PCC references than the national study program  and local program syllabuses . On the one hand, our results could be interpreted as a lack of governance from authorities and leading politicians regarding the national study program. There is also a lack of governance of the faculties and departments at the universities when it comes to the local program syllabuses. On the other hand, the results could be seen as a good example of an implementation process driven by a bottom-up process in which the local teachers are the ones taking lead on the change.We identified local intended learning outcomes with PCC in three of the four national study programs at level II, and all examined national study programs had intended learning outcomes with PCC in their local course syllabuses (level III). The implication is that changes in the four Swedish national study programs are driven more often by the university lecturer, given that the local course syllabuses  could be classified within the skills/ability heading. It is reasonable that HEIs promote skills/ability if person-centered ethics  is the tThe uneven representation and distribution of PCC between and within programs calls for a consistent implementation strategy. Implementation requires that several actors from different societal systems work together, take a unified approach, and mutually drive the change forward , 19, 20.Many factors affect the quality of research when reviewing documents , and thiThe change towards more PCC within the educational system is driven by local course leaders and teachers. Most PCC content found within the study programs was at a local level in intended course-learning outcomes. Increasing the inclusion of PCC instruction within and between the national study programs, local program syllabuses, and local course syllabuses requires action from politicians, authorities, faculty, and departments of higher education Fig. . There i


Based on the US DARE-kiR, a version of the Keepin’ it REAL program, the Drug and Violence Resistance Educational Program (PROERD) is the most widely implemented Brazilian prevention program. It originates from the translation of the DARE-kiR, a version of the Keepin’ it REAL program. Previous results suggest its inefficiency in preventing drug use among Brazilian adolescents. Since kiR fidelity can impact program outcomes, this mixed-methods study evaluates the PROERD implementation fidelity and its effects on preventing drug use among adolescents.Data from two cluster randomized controlled trials (cRCTs) with 4,030 students from 30 public schools in São Paulo , assessed at two-time points, were analyzed quantitatively. After implementing each lesson during the cRCT, 19 PROERD instructors answered fidelity forms. The effect of PROERD fidelity on alcohol, cigarettes, marijuana, inhalant, and cocaine use (the last two only among seventh graders) in the six months prior to follow-up assessment was analyzed by logistic regressions for fifth grade and mixed effect models for seventh graders. For qualitative analysis, semi-structured interviews were conducted with PROERD instructors and investigated by thematic analysis.Quantitative analysis showed that PROERD implementation fidelity had no impact on drug use among fifth and seventh graders. Conversely, the qualitative analysis revealed important aspects that may influence implementation fidelity and consequently program effectiveness, such as adaptations made by instructors, school infrastructure, among others, besides program application.PROERD requires cultural adaptation to improve its implementation in Brazilian public schools.The online version contains supplementary material available at 10.1186/s13011-022-00496-w. Adolescence is the typical period during which initial alcohol and drug use occurs , impactiPrograma Educacional de Resistência às Drogas e à Violência – PROERD) is the most widely implemented school-based prevention curricula, reaching approximately 40% of schools [Among Brazilian adolescents between 13 and 15 years of age, 55.5% reported alcohol consumption and 9.0% had used illicit drugs at least once in their lives, whereas 22% reported episodes of drunkenness . Despite schools . A recen schools .Its current curriculum is a Brazilian Portuguese translation of the US DARE-Keepin’it REAL (DARE-kiR) program , renamedConsidering these unexpected results from the Brazilian version of the DARE-kiR program, we must investigate which factors might be affecting the outcomes. One factor that reportedly impacts a prevention program’s effectiveness is the excellence (or lack thereof) of its implementation , which cAccording to Pettigrew et al. , teacherGiven the relevance of implementation fidelity to better program outcomes, we hypothesized that PROERD delivered with high fidelity would have a better effect among students. Hence, this study sought to evaluate, by quantitative and qualitative methods, the PROERD implementation fidelity and its effect on drug use prevention.Of a mixed-methods design, the study obtained quantitative data from two cluster randomized controlled trials (cRCTs) and collected qualitative data using (1) questionnaires applied to cRCTs participants, (2) fidelity forms answered by instructors (police officers) after each cRCT lesson, and (3) semi-structured interviews conducted with the instructors who delivered lessons. The interviews and qualitative analysis allowed us to answer research questions and clarify cRCT findings and quantitative results .The school-based “PROERD-Caindo na Real” program consists of 10 weekly classes (50 min each) delivered by trained police officers in the classroom environment, using student and teacher manuals. The police officer responsible for teaching the class uses the teacher’s manual, which provides information on procedures, objectives, materials needed, and tips for each lesson, including 1–3 activities. All participating officers underwent 80 h of training offered by the Military Police under the guidance of DARE America. Fifth and seventh graders were taught by the same instructor. All curricula were developed based on theories of narrative engagement , the priTwo parallel two-arm cRCTs were conducted with 4,030 fifth and seventh graders from 30 public schools in São Paulo in 2019 to evaluate the PROERD curricula for drug use prevention. Of these, 1,727 were fifth graders enrolled in 72 classes at 28 schools, and 2,303 were seventh graders enrolled in 90 classes at 30 schools. Both intervention groups attended 10 classes taught by 19 trained police officers; the control group received no intervention. State schools in the municipality of São Paulo that offered 5th and 7th grade and had not received PROERD in the last three years were included in the randomization. The first 30 schools listed were considered the study sample and the following 29 schools were included as backup in case of refusal. In the schools that agreed to participate in the study, all 5th and 7th graders participated in the cRCT. Sample size calculation, school selection, and the randomization process were performed according to Valente & Sanchez [Data were collected at two-time points. Baseline assessment was conducted prior to program implementation between February and March 2019. As the Brazilian academic year usually runs from February to December, follow-up data were collected 9 months after baseline in November and December 2019. Control and intervention data were collected simultaneously. An anonymous, self-administered audio-guided questionnaire was applied to students using smartphones by researchers in the classroom without a teacher. This instrument has been employed in previous studies to evaluate school-based drug prevention programs in Brazil , 42. It The outcome analyzed was the prevalence of drug use among fifth and seventh graders in the past 6 months before the follow-up assessment (yes or no), including alcohol, tobacco, marijuana, binge drinking, inhalants, and cocaine (the last two assessed only among seventh graders). During follow-up, the adolescents answered questions such as “Have you drunk alcoholic beverages in the past six months?” Binge drinking was considered as the consumption of five or more alcoholic beverages on a single occasion.Control variables consisted of sex , age, and socioeconomic status (SES). SES was assessed by the Brazilian Association of Research Companies (ABEP) scale, which considers the schooling level of the head of the household and the goods and services used. ABEP score ranges from 1 to 100 points, graded from A (highest) to D/E (lowest) according to established cutoff points: A (45–100 points), B (29-44 points), C (17-28 points), and D/E (0–16) .In each assessment, students provided a code generated from letters and numbers of their personal information, which allowed to match pre- and post-tests, ensuring anonymity and confidentiality, as used previously in drug prevention program evaluations . Since spercentage of activity completeness , and percentage of alterations . A fidelity variable for each class was calculated as follows: fidelity = % completeness× . Classes were then divided into two groups according to the level of fidelity: those that received ≥ 80% of the proposed PROERD activities were considered to have completed the program, whereas those that received ˂80% of the activities were considered to have incomplete implementation.Data on implementation fidelity were collected using self-administered online questionnaires completed by the instructors after each lesson, reporting whether the scheduled activities were delivered (yes or no) and whether any activities were altered by the instructors (yes or no). The fidelity forms listed all activities planned for each program lesson, based on the teacher’s manual. The first author (J.D.G.) trained the police officers on how to fill the form. Each item had data on the Since we evaluated 35 fifth grades and 47 seventh grades, and awaited 10 fidelity forms from each class (1 for each of the 10 lessons delivered), we expected a total of 350 and 470 forms for fifth and seventh grade classes, respectively. However, some instructors failed to return the forms, resulting in 47 (13%) and 83  fifth and seventh grade forms not delivered and considered missing. Online completion allowed researchers to mark all responses as “mandatory,” thus avoiding missing answers. Completeness and alteration calculations were proportional to the information available without missing data.After the intervention, data were collected by semi-structured interviews conducted with all 19 PROERD instructors involved in the cRCT (19 instructors delivered the program in 30 intervention schools).Qualitative data were collected by semi-structured in-depth interviews , with a Interviews lasted around 45 min and touched on the following topics: (i) how and why the participants became a PROERD instructor; (ii) how they perceive the effects (if any) of the program; (iii) their relationship with the school counselor and how it could affect the program’s impact; (iv) whether they considered the program training sufficient and how it influenced their work in PROERD; (vi) their opinions on the material and its content; (vii) what planned activities were well and poorly received; (viii) differences in applying the curricula for fifth and seventh graders; (ix) how PROERD demands affected other officer duties and vice versa; (x) what they would change in the program; (xi) what could be done to improve PROERD implementation. All interviews were recorded with prior consent from the interviewees. The resulting data were anonymized, transcribed verbatim, and identified by an alphanumeric code combining the letter (P), for police officer, and a random number assigned according to the order of the interviews .Sociodemographic characteristics and drug use data underwent descriptive analysis, with categorical variables expressed as numbers and percentages, and numerical variables expressed as means and standard deviations. We then performed inferential analysis to assess the impact of PROERD implementation fidelity on reducing adolescent drug use. For seventh graders, the impact of PROERD implementation fidelity on alcohol, cigarette, marijuana, inhalants, and cocaine use and binge drinking in the past 6 months was examined by a mixed-effects linear model. This model considers variability within individuals (from baseline to follow-up) and between individuals (children nested in schools), highlighting the relations between the observed response and explanatory covariates , 51. GivData obtained from the fidelity forms on the completeness and alterations of PROERD lessons were underwent descriptive analysis, summarized as numbers and percentages.Data underwent thematic analysis  using axAfter initial coding performed by the first author (J.D.G.), a PhD candidate in public health with training and experience in qualitative analysis, data underwent interpretive triangulation by the second author (R.G.C.), an anthropologist and PhD candidate in collective health, who analyzed the data in parallel. Disagreements were resolved by discussions, and a second review of the interview transcripts. Topics reported and discussed in this manuscript resulted from a consensus among researchers.Among fifth graders, 1,727 students answered the baseline questionnaire and 1,334 the follow-up questionnaire (77.24%). Among seventh graders, 2,303 students answered the baseline questionnaire and 1,739 the follow-up questionnaire (75.51%) . We observed no effect of implementation fidelity on reducing drug use for either grade  being the most incomplete (20% completeness) and lesson three (making choices) the most altered . Similarly, 61.7% of seventh grade classes received the program with high fidelity, with lesson ten (eco map) being the most incomplete (67.5% completeness) and lesson four  the most altered  Table S3 (Supplementary File) summarizes the interviewees’ characteristics, and Annex S1 (Supplementary File) presents the semi-structured interview script applied to police officers.Findings were classified into three dimensions—adaptations, accumulation of functions, and school infrastructure—, corresponding to the eight codes. Table Adaptations: Of the 19 interviewed instructors, 15 reported adapting the program, usually spontaneously and based on individual perception of student’s needs (without any systematics), such as reading and writing difficulties. Instructors also chose to adapt the program when lessons were thought to flow better if applied differently from the established program. We identified four types of adaptations to PROERD curricula: alterations according to student literacy, addition of content and/or activities, changes in curricula, and exclusion of content and/or activities.Accumulation of functions: Accumulation of two functions strongly influenced the instructor’s work. Police officers responsible for implementing PROERD do not necessarily leave their regular policing duties, which hinders the planned 10-week application, as some officers may need to report for duty during class hours or because their shifts interfere with class preparation, thus impacting the intended program delivery. Only four instructors reported being rarely assigned to other activities besides the program, which facilitated their work as PROERD instructors. Of the remaining interviewees, 11 stated being assigned to other activities or experiencing a lack of support from their superiors, as they eventually missed classes due to other duties.School infrastructure: According to the instructors, the reality of public schools in São Paulo makes it impossible to show videos included in the program’s material, resulting in adaptations to the curriculum.This study analyzed the impact of PROERD implementation fidelity based on quantitative and qualitative methods. According to quantitative results, the level of implementation fidelity had no influence on the program’s ability to reduce adolescent drug use. Conversely, the qualitative analysis revealed important implementation aspects that must be considered when examining the program outcomes, such as adaptations made by the instructors, accumulation of functions , and school infrastructure.Contrary to previous literature, which suggested that implementation fidelity plays an important role in program outcomes , 30, theThe higher rates of low implementation fidelity suggest that instructors, when implementing evidence-based programs in schools, often encounter unpredictable situations that lead to changes in activities, such as lack of school infrastructure, affecting the level of implementation fidelity , 33. DifQualitative data revealed that instructors adapted the program curricula according to aspects such as student literacy, school infrastructure, and previous teaching experience, thus compromising implementation fidelity. Previous studies , 61 haveSurvey on the Use of Information and Communication Technologies in Brazilian Schools [According to the  Schools , most pu Schools  state thAccording to Medeiros et al. , teacherIn analyzing the interviews, it became evident that the null effect of implementation fidelity lies on the lack of cultural adaptation, as officers reported having to adapt the program to make implementation feasible due to student difficulties and school infrastructure.A first limitation to consider is that the schools selected to participate in the RCTs were located in the low-income regions of São Paulo, which experience high exposure to drug use ; hence, This study is the first to evaluate PROERD implementation fidelity in Brazil, highlighting important considerations to improve is effectiveness and sustainability. Future studies examining these parameters should employ better reliable instruments to measure implementation fidelity , such as class observations. Moreover, future PROERD investigations must define which activity touches upon the core element of a lesson . PROERD Below is the link to the electronic supplementary material.Supplementary Material 1


Loyalty has always been a hot topic in the field of organizational behavior, which is of great significance to the operation and management of organizations. Extant studies have found that leadership can have a great impact on loyalty. Differential leadership is a common and unique leadership style in Chinese organizations, but the research between differential leadership and loyalty is still lacking. Based on social exchange theory and social learning theory. This study discusses the relationship between differential leadership and loyalty. By using hierarchical regression analysis and bootstrap method. The results show that differential leadership has a positive effect on employees’ organizational loyalty and supervisory loyalty, and the degree of “loyalty to organizations” is generally lower than “loyalty to supervisors”; under differential leadership style, the supervisory loyalty from insiders is much higher than that from outsiders, but the influence and difference of the two groups (insider and outsider) do not have a significant effect on organizational loyalty. The supervisor developmental feedback mediates the relationship between differential leadership and organizational & supervisory loyalty; power distance positively moderates the relationship between supervisor developmental feedback and organizational & supervisory loyalty, and the higher the perceived power distance of employees, the stronger the indirect effect of developmental feedback from supervisors. This study not only enriches the theory of differential leadership but also provides empirical support for understanding the differences in cognitive behavior between insiders and outsiders. The levCurrent research on loyalty focuses on sustainability actions, organizational justice, and organizational culture . DiffereAccording to differential leadership theory, leaders divide their employees into insiders and outsiders based on three factors: “Kinship, loyalty and ability” . On the The differential leadership treats insiders and outsiders differently, which inevitably leads to different reflections on employees’ psychology and behavior. So, what is the process mechanism of this influence? Scholars such as In addition, the study of employee loyalty behavior in the Chinese context cannot ignore the cultural value orientation of the employee . As a tyFrom the Soil: the foundations of Chinese society. That the relationship (guanxi) structure of Chinese society shows a self-centered pattern of differential treatment based on the closeness and remoteness of relationships with others, which echoes the “differential and order of human relationships (ren lun)” mentioned in the Book of Rites, the “differential” means “intimacy” (qin qin), which refers to the horizontal relationship of closeness and remoteness based on blood or marriage, such as the relationships ; “order” means “respect” (zun zun), which reflects the vertical relationship of superiority and inferiority based on rights and obligations, such as the relationship (monarch and subject). As a typical social psychological characteristic of Chinese society, the differential pattern exists not only embodies the macroscopic social aspect, but also the middle organizational aspect and the microcosmic interpersonal aspect (Fei (1992) pointed out in his book of l aspect , differel aspect . The funUnder the differential pattern, there is a tripartite interaction mechanism in the formation of behavioral loyalty. From the supervisor’s point of view, they will only give partiality and care to employees (insiders) from the employee’s point of view, the supervisor has the power to control important resources in the organization, which is extremely important to employee’s career development . AccordiOn the other hand, the identities of “insider” and “outsider” are not static, but even interchangeable under certain conditions . The difThe function of Loyalty is more like the “threshold” for employees to become an insider from an outsider, and meanwhile, loyalty is the first and most easily behavior for outsiders to imitate.To sum up, differential leadership is the core to decision-making, while providing scarce resources and development convenience for the insider, on the one hand, it strengthens the employee loyalty and identification to the supervisor, and meanwhile, strengthens the cohesiveness of the loyalty atmosphere for the organization. On the other hand, it attracts and promotes outsiders to show their loyalty to the supervisors in order to become an insider, which expands the radiating power of the loyalty atmosphere for the organization. In the process of exchange between the supervisor and the insider, and between the insider and the outsider  to 7 (completely agree).α = 0.903). For example, “When encountering conflicting views, I can clearly stand on the side of my superior (leader).”The measurement of this variable adopts the scale developed by α = 0.864), such as “My supervisor (leader) provides feedback to me mainly to help me learn and improve.” The Cronbach α coefficient of the scale is 0.864.The measurement of this variable adopts the scale developed by α = 0.907), such as “My organization gives me a sense of belonging.” Learned from the literature of relevant scholars like The measurement of this variable adopts the scale developed by α = 0.910). The scale includes five dimensions: internalization of leadership values, identification with leadership, dedication to leadership, additional efforts to leadership, and attachment to leadership.The measurement of Loyalty to Supervisors adopts the scale compiled by α = 0.933), including eight dimensions: internalization obligations, identification with the company, sacrifice for the public, advice and suggestions, assistance to colleagues, active participation, maintenance of public interests, and cooperation and compliance.The measurement of this variable adopts the scale developed by α = 0.831), such as: “leadership decisions do not need to consult the views of employees.”The measurement of this variable adopts the scale developed by We controlled for an assortment of variables, including age, gender, rank, working time with direct leaders, and current organizational working time are used as control variables.χ2/df = 2.918, TLI = 0.840, CFI = 0.852, GFI = 0.841, NFI = 0.847, RMSEA = 0.068, ∆χ2 = 265.426，∆ df = 4, p < 0.001) basically meets the requirements. The factor loadings of all indicators of their constructs ranging from 0.517 to 0.891 are significant at the 0.001 level. Compared to the four-factor model , three-factor model , two-factor model , and single-factor model , this indicates that the discriminative validity among the five variables in this study is good, which can be used for further data analysis.In this paper, confirmatory factor analysis is used to test the discriminant validity of differential leadership, superior developmental feedback, Power distance, organizational loyalty, and leadership loyalty. The results are shown in The effect of common method bias can be reduced through process control and statistical control. In this paper, privacy and confidentiality were emphasized in data collection to reduce the psychological concerns of the subjects, and the questionnaire was distributed at a series of different time to reduce the effect of common method bias through procedural control, but for the sake of scientific and rigorous research findings, the common method bias test was conducted by introducing the common method factor in the validation factor analysis The results are shown in r = 0.484, p < 0.001), power distance , organizational loyalty , and supervisory loyalty , and supervisor developmental feedback was also significantly and positively correlated with organizational loyalty ), supervisory loyalty , and power distance . The relationships between the variables were consistent with theoretical expectations and provided preliminary evidence for subsequent hypothesis testing.The means, standard deviations, correlation coefficients, and significance levels of each variable are shown in SPSS22.0 was used for hierarchical regression analysis in this study to, respectively, test the hypotheses of the overall sample , the sample of insiders , and the sample of outsiders .β = 0.447，p < 0.001)is greater than that of “loyalty to organizations” (β = 0.366，p < 0.001) after controlling for the five demographic variables of age, gender, rank, working time with supervisors, and organizational working time. The H1 was supported. Then, the “three-step approach” is used to test for mediating effects. From M2, it can be known that differential leadership has a significantly positive effect on supervisor developmental feedback (β = 0.486，p < 0.001). Next, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and organizational loyalty: It can be known from M5 that the regression coefficient of supervisor developmental feedback was 0.257 (p < 0.001), and the regression coefficient of differential leadership decreased to 0.241, but it was still significant at the level of 0.001, which indicated that supervisor developmental feedback had a partial mediating role in the relationship between differential leadership and organizational loyalty. Similarly, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and supervisory loyalty: it can be known from M10 that the regression coefficient of supervisor developmental feedback was 0.607 (p < 0.001), and the regression coefficient of differential leadership decreased to 0.152, but it was also significant at the level of 0.001, which indicated that supervisor developmental feedback had a partial mediating role in the relationship between differential leadership and supervisory loyalty. The H2 was supported.In this paper, a hierarchical linear regression model is used to test the direct effect and mediating effect proposed by the hypothesis, and the regression results are shown in In order to make the results more rigorous, used Model 4 in β = 0.141, p < 0.01) and , that is to say, power distance positively moderated the relationship between supervisor developmental feedback and organizational loyalty and supervisory loyalty. The significant interaction effect was further examined using simple slope analysis. When the dependent variable is loyalty to organizations, the association between power distance and supervisor developmental feedback was stronger for employees with high levels of power distance  than for those with low levels of power distance , and when the dependent variable is loyalty to supervisors, the association between power distance and supervisor developmental feedback was stronger for employees with high levels of power distance  than for those with low levels of power distance . H3 was supported.From M7 and M12 in In this paper, Model 14 in β = 0.550, p < 0.001)is greater than that of “loyalty to organizations” after controlling for the five demographic variables of age, gender, rank, working time with supervisors, and organizational working time. The H1 was supported.In this paper, a hierarchical linear regression model is used to test the direct effect and mediating effect proposed by the hypothesis, and the regression results are shown in β = 0.495, p < 0.001). Next, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and organizational loyalty: It can be known from M5 that the regression coefficient of supervisor developmental feedback was 0.168 (p < 0.001), and the regression coefficient of differential leadership decreased to 0.303, but it was still significant at the level of 0.001, which indicated that supervisor developmental feedback had a partial mediating role in the relationship between differential leadership and organizational loyalty; Similarly, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and supervisory loyalty: it can be known from M10 that the regression coefficient of supervisor developmental feedback was 0.507 (p < 0.001), and the regression coefficient of differential leadership decreased to 0.299, but it was also significant at the level of 0.001, which indicated that supervisor developmental feedback had a partial mediating role in the relationship between differential leadership and supervisory loyalty. The H2 was supported.Then, the “three-step approach” is used to test for mediating effects. From M2, it can be known that differential leadership has a significantly positive effect on supervisor developmental feedback  and , that is to say, power distance positively moderated the relationship between supervisor developmental feedback and organizational loyalty and supervisory loyalty. The significant interaction effect was further examined using simple slope analysis. When the dependent variable is loyalty to organizations, the association between power distance and supervisor developmental feedback was stronger for employees with high levels of power distance  than for those with low levels of power distance , and When the dependent variable is loyalty to supervisors, the association between power distance and supervisor developmental feedback was stronger for employees with high levels of power distance  than for those with low levels of power distance . H3 was supported.From M7 and M12 in In this paper, Model 14 in β = 0.246，p < 0.001)is greater than that of “loyalty to organizations”(β = 0.388，p < 0.001) after controlling for the five demographic variables of age, gender, rank, working time with supervisors, and organizational working time. The H1 was supported. Then, the “three-step approach” is used to test for mediating effects. From M2, it can be known that differential leadership has a significantly positive effect on supervisor developmental feedback (β = 0.366，p < 0.001). Next, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and organizational loyalty: It can be known from M5 that the regression coefficient of supervisor developmental feedback was0.569(p < 0.001), and the regression coefficient of differential leadership decreased to 0.180, but it was still significant at the level of 0.001, which indicated that supervisor developmental feedback had a partial mediating role in the relationship between differential leadership and organizational loyalty. Similarly, the variation of supervisor developmental feedback was added to the regression equation of differential leadership and supervisory loyalty: it can be known from M10 that the regression coefficient of supervisor developmental feedback was 0.650 (p < 0.001), and the regression coefficient of differential leadership decreased to 0.008, which is no longer significant, which indicates that superior developmental feedback plays a completely mediating role in the relationship between differential leadership and loyalty to supervisors, that is, the effect of differential leadership on loyalty to supervisors is completely realized through superior developmental feedback. The H2 was supported.In this paper, a hierarchical linear regression model is used to test the direct effect and mediating effect proposed by the hypothesis, and the regression results are shown in In order to make the results more rigorous, used Model 4 in β = 0.174, p < 0.001). That is to say, power distance positively moderated the relationship between supervisor developmental feedback and organizational loyalty. But for loyalty to supervisors, the interaction effect was not significant . The significant interaction effect was further examined using simple slope analysis, the association between power distance and supervisor developmental feedback was stronger for employees with high levels of power distance  than for those with low levels of power distance . H3 was partially supported.From M7 and M12 in In this paper, Model 14 in Unlike previous studies that focused on the relationship between differential leadership and organizational citizenship behavior, this paper focuses on the types of employee loyalty and constructs a theoretical model of differential leadership and organizational loyalty and leadership loyalty in the context of Chinese culture, and finally conducts an empirical analysis from a perspective of insider and outsider on the basis of social exchange theory and social learning theory. Research indicates:First, differential leadership has a positive effect on employees’ organizational loyalty and supervisory loyalty. No matter in the overall samples or the sample of insiders, the positive effect of differential leadership on employees’ supervisory loyalty is significantly higher than that of differential leadership on organizational loyalty. However, in the samples of outsiders, the positive effect of differential leadership on employees’ supervisory loyalty was lower than that on organizational loyalty.Second, the effects of differential leadership on supervisory loyalty of insiders and outsiders are significantly different. Specifically, the regression coefficient of differential leadership on supervisory loyalty of outsiders is 0.246 and that of differential leadership on supervisory loyalty of insiders is 0.550, but the disparity between the effects of differential leadership on organizational loyalty from insiders and outsiders is not significant.Third, supervisory developmental feedback mediates the relationship between differential leadership and organizational and supervisory loyalty.Fourth, power distance positively moderates the relationship between supervisory developmental feedback and organizational and leadership loyalty, and the higher the employee perceives power distance, the stronger the indirect effect of supervisor developmental feedback on employees’ organizational and supervisory loyalty is.First, differential leadership, as a common and unique leadership style in Chinese organizations, has not been paid much attention by academia. This study confirms the relationship between differential leadership and employee loyalty. The positive influence of differential leadership on employee loyalty could be explained from the two aspects of “reciprocal” (bao) theory and social learning theory. On the one hand, the “reciprocal” (bao) culture has a profound historical origin in the Chinese traditional culture, and “return a favor with a favor” has always been the mainstream value respected by the Chinese nation, so when the insiders receive the partiality from differential leadership , they make good psychological contracts and show great loyalty to their supervisors; on the other hand, the partiality from differential leadership is much like a booster for employee’s career development and life goals and is equally important for outsiders. So if the outsider wants to receive the same treatment, they must become an insider from an outsider, according to the alternative reinforcement mechanism of the social learning theory, loyalty to supervisors is one of the best ways. In the three-way game between the leader and the insider and the outsider, the insiders will show greater loyalty to the supervisor because they are favored; while the outsiders will want to become an insider because they are not favored. This game process continuously strengthens the cohesion and radiation of the “influence,” and also enhances employee loyalty. This study selects the differential leadership with local cultural adaptability and characteristics as a breakthrough, which not only enriches the theory of local leadership but also is a useful supplement to the influencing factors of loyalty behavior.Second, the study on the influence of differential leadership and loyalty behavior from a perspective of insiders and outsiders broke the logical limitations of previous studies from a single perspective and conducted separate tests for the samples of insiders and outsiders as well as the overall test, which not only improved the accuracy of the test results but also provided a detailed data comparison for the differences of the results. In the first place, it was found that the regression coefficient of supervisory loyalty was significantly larger than that of organizational loyalty in the sample of insiders, that is to say, differential leadership has a more significant effect on the formation of “loyalty to supervisors,” but in the sample outsiders, the regression coefficient of leadership loyalty was smaller than that of organizational loyalty, which may be due to the fact that outsiders lack of psychological attachment to their direct supervisors compared with the insiders. They only maintain an instrumental relationship with their leaders that only involves the equal exchange of benefits and harms . The lower level of emotional exchange between employees and supervisors makes outsiders strongly believe that there is no significant difference in the concept and connotation of “organization” and “leadership,” and unconsciously regard the supervisor developmental feedback as care from the organization, instead of a sort of partiality from supervisors. Therefore, the regression coefficient of employee loyalty to the organization of outsider is slightly higher than that of loyalty to supervisors; next, It can be found that the regression coefficients of differential leadership on the formation of supervisory loyalty of insiders is much higher than that of outsiders, that is to say, employee loyalty to supervisory of insiders is significantly obvious than that of outsiders. This is consistent with the multifocal perspective and the object consistency model in organizational behavior research: the receiver’s response is directly dependent on the specific object that releases the signal. After differential leadership releases a signal of partiality, it stimulates insiders to show positive behaviors of loyalty to the supervisor. Although the signal also stimulates outsiders to become insiders by showing their loyalty to the supervisor, the degree of loyalty of outsiders is definitely not as high as that of insiders. This study examines differential leadership and loyalty behavior from a perspective of insider and outsider, which is not only supplement and extension of existing single perspective studies but also provides empirical support for understanding the differences in cognitive behavior between insiders and outsiders.Third, this study further reveals the process mechanism of the influence of differential leadership on employee loyalty, verifies the mediating role of supervisor developmental feedback, analyzes an important inducing factor of employee loyalty, and to some extent exposes the “black box” (an intermediate mechanism) of differential leadership influence on employee loyalty.Fourth, from the perspective of employee traits, this study examines the moderating role of power distance in the formation of employee loyalty, and supplements the boundary conditions for employee loyalty behavior. As a country with a high power distance, people in China generally respect authority and are very sensitive to the behavior of authority figures. Employees who live in a high power culture are more likely to return a psychological favor after receiving developmental feedback from their supervisors and then behave in a loyal way to their supervisors and organizations. This suggests that the formation of employee loyalty not only depends on leadership style but also on employee traits to some extent, so the two factors cannot be generalized and confusing. This study selects power distance as a moderator when examining the mechanism of differential leadership on employee loyalty, and finds that outsiders feel a sense of supervisor unfairness and organization alienation, the result is that outsiders could not make the psychological contract with their supervisors. Therefore, outsiders have unclearer thoughts of the organization and their immediate supervisor than insiders, and even generally regard the two as the same concept. So the moderating effect of power distance on the formation of leadership loyalty of outsiders is not obvious, but the moderating effect in other samples is significant. This study examines the moderating effect from a perspective of insiders and outsiders, further clarifies the boundary conditions for differential leadership to be effective under different conditions, and provides a more detailed classification for the formation of loyalty behavior.First of all, leaders should take emphasis on their own management style and provide a fair and reasonable channel for outsiders to become an insider. While giving support and feedback to the insider, leaders should also actively communicate with outsiders, create a harmonious organizational atmosphere, improve the relationship between superiors and subordinates, in order to attract more outsiders, maximize the effectiveness of leadership, and constantly improve the overall loyalty of the organization.Secondly, leaders can actively implement the strategy of supervisor developmental feedback, which is the intermediate path to stimulate and promote employee loyalty behavior. Leaders should give full play to their initiative and actively communicate this feedback to employees in an appropriate manner and with the right frequency. At the same time, they should enhance their learning on feedback and improve its way and quality so that Employees could feel the care and partiality of their supervisors, thus improving employee loyalty.Finally, Perceived power distance of employees could moderate the effect of differential leadership on employees’ loyalty behavior. In the practical management process, leaders should implement differential management according to the level of perceived power distance of employees. Especially for those employees with a high power distance, they are more likely to return psychological favor after receiving positive feedback from supervisors. Therefore, leaders should pay more attention to communicating with those high power distance employees reasonably and effectively in order to maximize their employee loyalty.Although this paper uses a multi-period method to distribute the questionnaires, since the questionnaires are filled out by the participants in the form of self-assessment, even if the problem of common method bias is not serious, the problem cannot be completely eliminated. In the future, the sources of other evaluation data should be increased to reduce the common method bias. In addition, this study confirmed that differential leadership has a positive effect on employee loyalty, but in general, such loyalty can be classified as “loyalty to supervisors.” However, on the individual level, there is no doubt that loyalty to supervisors could reduce and avoid conflicts between superiors and subordinates and thereby improve the management efficiency of the leader, but at the organization-wide level, overemphasizing leadership loyalty will cause factions within the organization, which in turn affects the overall efficiency of the organization. Therefore, future research should further explore how to achieve the integration of “loyalty to organizations” and “loyalty to supervisors” in order to maximize the overall effectiveness of the organization.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Yunnan University of Finance and Economics. The patients/participants provided their written informed consent to participate in this study.HZ: supervision and funding acquisition. LD: conceptualization, methodology, formal analysis, and writing—original draft. ZJ: editing. All authors contributed to the article and approved the submitted version.This study is supported by the National Natural Science Foundation of China “Research on the Causes, Evolution and Resolution Mechanism of Ought to be Conflicts in Mixed Ownership Reform: The Perspective of mutual beneficial organization”. (Grant No. 72262035).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: The giant gourami sago strain (Osphronemus goramy Lacepède) has been approved in 2018 as a candidate for freshwater aquaculture in Indonesia. However, information on the species’ reproduction is minimal. This study analyzed the reproductive characteristics of the gourami sago strain broodfish to provide basic knowledge for a future hatchery development strategy.Methods: A total of 10 female and 10 male mature gourami sago strain broodfish were measured for body weight and length, and were evaluated for their reproductive characteristics.  Breeding fish were spawned naturally in a 2×1×0.6 m concrete pond with a male-female sex ratio of 1:1. Egg weight and diameter were measured in 25 eggs per female using, respectively, ACIS AD- 600i scales with 0.01 g accuracy, and a microscope (Labo model L-711) using Canon Digital Camera Software 3 . Semen was collected using plastic syringes in 3 mL aliquots, then placed in an insulated ice-cooled container, and analyzed within two hours of collection.Results: Average weights of female and male broodfish before spawning were 2180±159.78 g and 3060±134.99 g, respectively. The relative fecundity and egg diameter were 1029±36 eggs kg-1 and 2.42±0.05 mm, respectively. The hatching rate and embryo survival to an eyed-egg stage were respectively 76.40±2.27% and 94.76±0.42%.  Sperm characteristics showed that volume was 0.60±0.12 ml kg-1 and percentage of motile sperm was 70.04±2.27%. Female broodfish weight after spawning was strongly correlated with the weight before spawned (r2 = 0.999) and absolute fecundity was also strongly correlated with female broodfish weight before spawning (r2= 0.921). Sperm concentration was moderately correlated with sperm motility (r2 = 0.556) and duration of sperm motility (r2 = 0.502).Conclusions: The gourami sago strain broodfish has suitable reproductive characteristics for the development of hatcheries. Successful natural spawning should be followed by larval weaning and feeding technology to increase growth and survival. Approximately 77.57% of fish produced in freshwater aquaculture in Indonesia are sourced from freshwater ponds and inland waters. However, their development depends upon many factors, such as fish species, aquaculture systems, water depletion, fish diseases, farmers’ knowledge, and aquaculture practices.Freshwater aquaculture practiced in inland waters such as lakes, rivers, reservoirs, floodplains, oxbow lakes, and freshwater ponds have expanded during the last decades in Indonesia. Nile tilapia (Oreochromis niloticus) contributed 37.93% of the total aquaculture production, African catfish (Clarias gariepinus) 33.35%,Pangasius catfish  12.38%, common carp (Cyprinus carpio) 9.28%, and giant gourami (Osphronemus goramy) 6.96%.Freshwater aquaculture is one of the fastest-growing aquacultures in Indonesia, with more than 3,378,298.92 metric tons produced in 2018. However, they have not been able to contribute majorly to freshwater aquaculture production in Indonesia. Although the gourami sago strain has been approved as a candidate for aquaculture production, its distribution is limited in the West Sumatra Province of Indonesia. Therefore the development of its hatchery is necessary to be able to contribute to the production of freshwater aquaculture. The gourami sago strain is considered to support food security along with many other freshwater fish species in Indonesia.Indonesian giant gourami strains include the local "tambago”, “palapa”, “soang”, “galunggung” and “blusafir" strains, which have been grown semi-intensively in small-scale farms for decades. The gourami sago strain is an native strain in Lima Puluh Kota Regency, West Sumatra Province. In this strain, the head, caudal and dorsal fins are yellowish red. The morphometric character between the sago gourami strain and the different palapa, tambago, krista, and jepun strains is the distance between the base of the dorsal fin to the tip of the anal fin and the lower part of the pelvic fin is higher. However, data on its reproductive characteristics are still limited. The evaluation of reproductive performance in other fish species has had beneficial impacts in the development of freshwater aquaculture in Asia. In contrast, there are still gaps in knowledge of giant gourami sago strain broodfish regarding size at oocyte maturity, age of sexual maturity, sperm characteristics, egg hatchability, survival after eyed-egg stage, larva weaning, and growth rate. These factors were identified as key challenges for successful giant gourami sago strain hatchery performance in the future. Therefore, the present study was conducted to evaluate the reproductive characteristics in the giant gourami sago strain to provide basic knowledge for a hatchery development in the future.The gourami sago strain was approved as a candidate for freshwater aquaculture in 2018 .Osphronemus goramy) as a candidate for future aquaculture. The study was funded by the Research and Community Service, Universitas Bung Hatta under a competitive grants scheme called the research of Professor in 2021 (Contract number: 06.02.1.46.03.2021). This grant included ethical approval and permits to collect fish specimens, rear and spawn giant gourami sago strains in the Aquaculture Laboratory Faculty of Fisheries and Marine Science, Universitas Bung Hatta facilities. There was no animal suffering involved in this study and gourami sago strain broodfish were still in good condition when returned to the pond. Ethical approval was granted by the Ethics Commission for Research and Community service at Universitas Bung Hatta (023/LPPM/Hatta/I-2021).There are no required permits from the government of the Republic of Indonesia to evaluate reproductive characteristics in gourami sago strain broodfish  concrete freshwater pond. During the rearing of the fishes to sexual maturity, the juvenile fish were fed with commercial fish pellets .3 (6 × 2 × 1.5 m) concrete freshwater ponds for 60 days. The broodfish were fed twice daily (09:00 AM and 16:00), with extruded feed pellets containing 39.50% crude protein and 12.21% fat with a predetermined quantity of 3% of fish weight per day. Besides that, the fish were given sente leaves (Alocasia macrorriza L.) at amounts of 1% of fish wet weight per day. The leaves contained 2.85% protein and 0.47% crude fat (of wet weight). Each concrete freshwater pond had a 50 mm drain in the middle, which was covered with a net of 0.5 cm mesh size to prevent the fish from escaping and predators from entering. Water was pumped from borehole wells at a rate of 5 liters per minute.After the giant gourami sago fish were reared for five years, 20 mature individual broodfish were separated according to sex and reared in two 18 mA total of 20 broodfish with mature oocytes were selected, consisting of 10 females and 10 males. Before spawning, female and male broodfish were weighed using scales (OHAUS model CT 6000-USA with 0.1 g accuracy), and body length was measured using a meter ruler with 1mm accuracy. The average weight and length of the 10 female broodfish were 2140 ± 159 g and 39.70 ± 1.77 cm, while those of the male broodfish were 3060 ± 135 g and 43.1 ± 1.79 cm.•3 × 100Condition factor (CF) = wet weight in gram/length•Ovulated egg weight (g) = fish weight before spawning (g) – fish weight after spawning (g)•Ova somatic index (%) = egg weight ovulation (g)/fish weight before spawning (g) × 100Reproductive parameters in gourami sago strain broodfish were analyzed using the following formulae:Absolute fecundity was the total number of eggs estimated per nest, and relative fecundity was the total number of eggs per kg body weight.−1), based on the dosage used forHemibagrus wyckii. Oocyte maturation was assessed for each individual. The oocyte maturity in giant gourami females was assessed from oocytes sampled by intraovarian biopsies using a flexible polyethylene catheter. Egg diameter was measured using Labo microscope model L-711 andCanon Digital Camera Software 3.Starting in August 2020 onwards, the broodfish were checked monthly for eggs and semen production. The broodfish were captured with a hand net and anesthetized by oral ingestion of Tricaine methanesulfonate  concrete freshwater ponds with a male-female sex ratio of 1:1. Before the broodfish spawned, the ponds were drained, cleaned, and all other species removed. Then, palm fibers were placed on top of a bamboo raft in the pond. The pond was then filled with water, and the female and male broodfish were released into the spawning pond. The male broodfish made a nest for five to seven days, after which spawning took place and the female broodfish laid eggs. Spawning occurred in the afternoon (between 3.00 to 5.00 PM). Due to the presence of a very large oil globule, giant gourami eggs float. After the broodfish had finished laying eggs, the eggs were kept by the female broodfish in the nest. After the eggs had been kept by the broodfish for four hours in the nest, the eggs were collected and transferred to an incubation tray, which was placed in a ventricular hatching system. A total of 100 eggs for each broodfish were incubated in incubation trays. Meanwhile, the broodfish were returned to their pond once spawned, and no mortality occurred.Natural spawning of broodfish was carried out in 1.2 mCanon Digital Camera Software 3. A total of 25 eggs were randomly sampled 16 hours after spawning to determine the fertility rate (FR). The hatching rate (HR) was determined by counting all hatched fry 48 hours after spawning. Then, the endogenous feeding period of the fish larvae was counted until the egg yolks ran out (in days), and embryo survival rate (%) to the eyed-egg stage was measured.Egg weight and diameter were measured for 25 eggs per female using SHIMADZU-model AY 220 scales with 0.1 mg accuracy and a microscope (Labo model L-711) using1 of MS-222, then fish weights (MaW) and total lengths (MaL) were measured. Special care was taken to avoid any contamination of semen with urine, feces, mucus, and water. Semen samples were collected using plastic syringes in 3 mL aliquots and then placed in an insulated ice-cooled container, transported to the laboratory, and analyzed within two hours.To stimulate the spermiation process in male broodfish, an LHRH-preparation  was injected into male fish, with a dosage of 0.5 ml per kg of the brooder. Semen samples were obtained from 10 gourami sago broodfish randomly selected from the farm. The male broodfish were first anesthetized with 50 mg Lˉet al. and Cabritaet al. The gross examination was based on visual and physical observation of parameters like semen volume, semen pH, sperm concentration, motility, and duration motility. Semen volume was determined by collecting the semen in a graduated cylinder. Semen pH was determined with a hand pH meter . Microscopic examination was carried out using the Olympus model CX40, with × 10 and × 25 magnification, to determine parameters such as motility (MO) percentage and duration, by observing water-activated semen placed on a glass slide. Motile sperm were observed and expressed as a percent of non-moving sperm. Motility duration (DMO) was determined as the period between movements of the sperm to a cessation of any progress using Neubauer’s hemocytometer and calculated as the number of sperm ml−1. Semen pH was determined with a hand pH meter .The sperm assessment included gross  and microscopic examination as reviewed by Rurangwaet al., 2012. pH values were determined with a pH meter . An oxygen meter  was usedin situ, and water temperature in the spawning pond and incubation trays were measured with a thermometer .Water samples were collected in the spawning pond and incubation trays to determine alkalinity, hardness, and pH. The protocol for determining alkalinity by standard methodology is presented by RiceSPSS software . The standard deviation of each parameter was determined. For linear regression analyses, correlations were considered significant at p < 0.05, and trends or tendencies were considered significant at p < 0.05.Results were given as the mean values (± SD). Simple linear regression analyses were performed usingThe reproductive characteristics of female broodfish in sago giant gourami are summarized inReproductive characteristics for male broodfish and sperm samples are presented inr2) between variables of reproduction characterization parameters in sago strain of giant gourami females broodfish results are shown inr2 = 0.921) and female fish weight after spawning (r2 = 0.864). Similarly, results revealed significant correlations between egg diameter and hardened egg diameter (r2 = 0.833), and between egg diameter and percentage of the hardened diameter (r2 = 0.699). Meanwhile, egg diameter and fertility rate were moderately correlated (r2 = 0.568). In contrast, the egg diameter was not strongly related to absolute fecundity (r2 = 0.169) and relative fecundity (r2 = 0.096). On the other hand, the survival rate of larvae (10 days) also had strong correlations with the hatching rate (r2 = 0.998) and endogenous feeding period (r2 = 0.757).The linear correlation (r2) between variables of reproduction characterization parameters in sago giant gourami male broodfish are shown inr2 = 0.836), while semen volume (r2 = 0.521), semen pH (r2 = 0.521) and sperm concentration (r2 = 0.506) were moderately correlated with gonad weight. In contrast, the gonadal weight negatively correlated with sperm motility (r2 = 0.017) and duration of motility (r2 = 0.275). In addition, the sperm concentration was also moderately correlated with the sperm motility (r2= 0.556) and duration of motility (r2= 0.502).Linear correlation analysis results  ranged from 977 to 1071 eggs kg−1. Egg produced in kg fish−1 (RF) is thought to be more informative than absolute fecundity. RF in sago strain of giant gourami was smaller compared to those in galunggung strain, palapah strain, and blusafir strain. On the other hand, the difference in relative fecundity can also be related to differences in broodfish size and age used. Environmental factors such as rainfall also influenced the number of eggs per spawning in giant gourami broodfish, while the water temperature negatively related to the number of eggs per spawning. Furthermore, egg diameter in sago strain of giant gourami was found to be almost the same as other strains of giant gourami  and survival rate (10 days) (r2 = 0.998). According to Sinket al., the biochemical composition of broodfish eggs is strongly correlated with egg quality. In this study, we did not evaluate the biochemical composition of an egg, because the relationship between egg quality and biochemical composition is difficult to interpret.In this study, the hatching rate of the embryo in the sago strain of giant gourami was smaller than those of other strains of giant gourami. In addition, oocyte development and maturation are also regulated by locally acting paracrine and autocrine signaling. However, there is no information about the effects of such factors on oocyte development in giant gourami sago. Still, the extrusion feed enriched with vitamin E (d-α-tocopherol) at concentrations of 137.8, 238.05, 338.72 and 439.39 mg per kg feed affected markers of reproductive functions of giant gourami broodfish, such as the sexual maturity cycles, ovum somatic index, relative fecundity, and egg diameter.The keys regulator hormones of fish reproduction are gonadotropins, follicle-stimulating hormone, luteinizing hormone, and sex steroids.Xiphophorus helleri,Channa marulius, andIctalurus punctatus. Additionally, implantation of 17ß-estradiol has also improved the reproductive performance inHemibagrus nemurus. Currently, whether the increase in the protein level of feed and use of hormones can increase the reproductive potential in sago giant gourami is poorly understood. Therefore, we recommend the use of proteins in feed  and the addition of 17β-estradiol  to increase the reproductive potential of giant gourami sago in the future.Various efforts have been made by scientists to increase the reproductive performance of female broodfish, such as increasing dietary protein levels forHemibagrus wyckii (0.60 to 1.20 ml), but higher than those ofPterygoplichthys gibbiceps. It appears that the semen volume depends on fish species. Many factors influenced sperm quality and quantity such as genetic, physiological, spawning season, and environmental factors. On the other hand, improvements in feed nutrition of broodfish can increase gamete quality and semen volume. Commercial honey combined with 10% Dimethyl Sulfoxide (DMSO) was also shown to increase sperm motility. Synthetic hormones such as gonadotropin-releasing hormone analogs (GnRHa), with or without dopamine antagonist, and domperidone (Dom) effectively improve sperm quality. Nevertheless, the duration of motility of sperm was strongly correlated with the water quality in ponds. In this study, the water quality parameters in spawning ponds included alkalinity of 50.5 to 52.5 mg/L HCO3− and hardness of 65.5 to 67.5 mg/L CaCO3, a pH between 6.4 and 6.6, and a water temperature between 28 and 30oC. These water quality parameters were able to support the ability of the sperm to fertilize the egg.Average semen volume in giant gourami sago was lower (0.4 to 0.6 ml) than those ofGenypterus blacodes andEsox lucius. Sperm motility includes the percentage of motile sperm, straight-line velocity, curvilinear velocity, average path velocity, and linearity. In this study, we did not investigate those parameters. In addition, the percentage of motile sperm is influenced by the addition of extenders and cryoprotectants. However, sperm motility from fresh semen was slightly greater compared to cryopreserved semen fromEsox lucius. The fertility rate of eggs ranged from 76 and 84%; however, no significant correlation was detected between fertility rate and sperm parameters, such as semen volume, semen pH, motility, and duration of motility. Conversely, the sperm concentration was moderately correlated with sperm motility and duration of motility. The parameters commonly measured to assess sperm quality in brood were volume, density, and motility , including fertilizing capacity. In this study, we did not investigate the ionic composition of the semen, but this phenomenon could be related to the ionic composition of semen which might have a significant influence on sperm motility and duration of motility.The sperm motility in sago giant gourami ranged from 68 to 75%, and the duration of motility ranged from 43 to 61 sec. These results are consistent withThis research analyzed the reproductive characteristics of giant gourami sago strain broodfish reared in concrete freshwater ponds, in the Aquaculture Laboratory Faculty of Fisheries and Marine Science, Universitas Bung Hatta. Relative fecundities of the giant gourami sago strain broodfish ranged from 977 to 1071 eggs, and egg diameter ranged from 2.32 to 2.46 mm. Semen volume ranged from 0.4 to 0.7 ml per kg body weight and sperm motility was comprised between 68 and 75%. A strong linear relationship was observed between absolute fecundity and female fish weight before and after spawning. Similarly, a strong, positive correlation was observed between survival rate (10 days) and hatching rate. The sperm concentration was also moderately positively correlated with the motility and duration of sperm motility. Keys to increasing the reproduction performance in gourami sago strain fish depend on broodfish weight, relative fecundity, and hatching rates. Although data on the reproductive characteristics of gourami sago strain broodfish have been obtained, there are still knowledge gaps in feeding technologies and larval weaning during rearing. Therefore, for successful practices in hatcheries, further research is recommended to determine a proper feed formulation and the development of appropriate aquaculture systems.Osphronemus goramy Lacepède, 1801): basic knowledge for a hatchery development strategy for the future.Figshare: Reproduction characterization of the gurami sago  in sago strain of giant gourami broodfish-Table 3. Raw data of hardened egg diameter (mm) in sago strain of giant gourami broodfish-Table 4. Raw data of egg diameter increase (%) in sago strain of giant gourami broodfish-Table 5. Raw data of egg weight (mg) in sago strain of giant gourami broodfish-Table 6. Raw data of hardened egg weight (mg) in sago strain of giant gourami broodfish-Table 7. The data of egg weight increase (%) in sago strain of giant gourami broodfish-Table 8. The data of fertilization rate (%) in sago strain of giant gourami broodfish-Table 9. The data of hatching rate (%), endogenous feeding period (day), and embryo survival rate to the eyed-egg stage (%) and in sago strain gourami-Table 10. Male size, gonadal weight, and semen in sago strain of giant gourami broodfish-9/mL) in sago strain of giant gourami broodfishTable 11. Sperm concentration (10-Table 12. Sperm Motility (%) in sago strain of giant gourami broodfish-Table 13. Duration motility (sec) in sago strain of giant gourami broodfishThis project contains the following underlying data:Creative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of the The article explaining reproduction performances of the giant gurami sago strain was well written and recommended to index.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Fish genetics and reproductionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. In general, the article is quite good explaining the reproductive performances of gourami sago. However, the introduction needs to state the distribution of gourami strain sago in Indonesia, whether it is endemic or introduced in the region. There is less information on the general characteristics of sago compared to other strains. Table 5 should explain some specific conditions of breeding technology used in other strains.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Fish genetics and reproductionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. I have evaluated this article and found the description of the approach taken for this study to be very appropriate, and each description is very detailed. Comparisons between previous studies are also fully informed in this article. I strongly support the following step of indexing of this article for the sharing of information on the reproductive characteristics of the giant gurami sago. I only found that a few corrections to the abstract need to be made, wherein the method section and the brand name need to be deleted from this section.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Fish amoeba, protozoology, cell biologyI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.


In conclusion, QKI-6 could be used to develop novel strategies for the treatment of NSCLC.The RNA-binding protein quaking homolog 6 (QKI-6) is a tumor-suppressor gene in several cancers. However, its role in non-small cell lung cancer (NSCLC) is unclear. In this study, we aimed to determine the association between QKI-6 expression and survival and clinicopathological features in patients with NSCLC and identify the related mechanisms. Western blot and immunohistochemistry (IHC) were used to detect QKI-6 expression in NSCLC. The effect of QKI-6 on NSCLC cells was determined by overexpression and knockdown assays, and label-free quantitative proteomics and Western blot were used to identify the underlying mechanisms. Low QKI-6 expression level was positively correlated with poor overall survival in patients with NSCLC. Furthermore, QKI-6 overexpression inhibited NSCLC cell proliferation and migration and induced a block in the G0/G1 phase, and QKI-6 downregulation increased proliferation and migration. QKI-6 inhibited EMT processes  Lung cancer is the leading cause of cancer-related death worldwide and has a mortality rate of 18.4% . Currentvia EMT inhibition (Quaking homolog (QKI) is part of the RNA-binding signal transduction and activation of RNA (STAR) protein family . The thrhibition . QKI-5 ihibition , 14. HowThe protein disulfide isomerase (PDI) anterior gradient protein 2 (AGR2) is typically present in the endoplasmic reticulum (ER). AGR2 suppresses cancer cell proliferation and metastasis , 16. AGRHere, we aimed to determine the function of QKI-6 in NSCLC progression and elucidate the underlying mechanisms. Low QKI-6 expression was positively correlated with poor overall survival. Furthermore, QKI-6 inhibited cell proliferation, migration, and EMT processes by downregulating AGR2 in NSCLC. Our results provide novel insights into the role of QKI-6 in NSCLC, and QKI-6 may be used as a diagnostic and therapeutic target for NSCLC.NSCLC tissues and matched peritumoral tissues were collected from patients with NSCLC who underwent surgical resection in Tangdu Hospital affiliated with Air Force Military University . All the patients met the criteria of the WHO 2004 classification . Tumors 2.Human lung cancer cell lines (A549) were purchased from the Type Culture Collection . Cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and penicillin–streptomycin (Gibco) and incubated at 37°C with 5% COThe QKI expression pattern was studied using NSCLC tissue microarrays, which was purchased from Outdo Biotech Company . Immunohistochemistry (IHC) staining was performed using the QKI antibody  as previously described . IHC imaPlasmids containing QKI-6 cDNA and plasmids expressing negative control shRNA (sh-NC) and QKI-6 shRNA (sh-QKI-6) were produced , and the lentivirus was generated in 239T cells by Biowit Technologies .5 A549 cells were seeded per well in six-well plates (Corning) and grown to 75% confluence. After changing the culture medium, lentiviral particles containing QKI-6 cDNA, negative control, QKI-6 shRNA, or negative control shRNA were added. Cells were then cultured at 37°C with 5% CO2. The culture medium was removed and cells were isolated using 5 μg/ml puromycin for 24 h. Infection efficiency was evaluated by Western blot.To obtain stable QKI-6 overexpression or knockdown, 1 × 10Total cellular protein from the A549 cell lines, NSCLC, and peritumoral tissues was extracted using RIPA buffer and quantified using the BCA assay. Sixty micrograms of protein from each sample was resolved by 10% SDS-PAGE, transferred to PVDF membranes (Millipore), which were then blocked in 5% skim milk in TBST, and incubated with primary antibody overnight at 4°C. The primary antibodies used were as follows: QKI , epidermal growth factor receptor (EGFR) , AGR2 , E-cadherin , N-cadherin , p-SRC Y418 , and p-STAT3 Y705 . After washing with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h. Protein bands were visualized using a Tanon 5200 chemiluminescent system .3 cells per well) for 1 to 5 days. The cells were treated with 20 μl sterile CCK-8 reagent per well and cultured for 4 h at 37°C. Absorbance was measured at 450 nm.Cell Counting Kit-8  was used to assess cell proliferation. A549 cells after gene transfection were plated in 96-well plates  were plated in the top chamber of an 8.0-μm membrane (Corning) Transwell in 200 μl DMEM without FBS. The bottom chamber contained 700 μl DMEM with 20% FBS. After 24 h, the migrated cells were fixed and stained with 0.1% crystal violet. Cells in six fields were counted randomly.A 24-well Transwell chamber was used for the migration assay. Cells (1 × 105) cells were plated in six-well plates and cultured for 20 h. After vortexing in PBS, the cell samples were fixed with 70% ethanol for 1 h. The cells were isolated by centrifugation and resuspended in PBS. The samples were incubated with propidium iodide (PI)/RNase (Thermo) for 30 min in the dark at room temperature and detected using a FACS Calibur (BD).A549 . Label-free quantification and LC/MS proteomics analysis were performed by the Mhelix Biotech Company . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium he PRIDE  partner ® 7500 Sequence Detection System. Amplification conditions were set to 40 cycles at 95°C for 5 s and 65°C for 30 s.Trizol reagent (Invitrogen) was used to isolate total RNA. According to the instruction of the M-MLV assay kit (Invitrogen), RNA was reverse-transcribed into cDNA. The primer sequences were as follows: AGR2-Forward: 5′-TTGTCCTCCTCAATCTGGTT-3′; AGR2-Reverse: 5′-ATCGGCTCTAACTGTCAGAGA-3′; GAPDH-Forward: 5′-GGGAAACTGTGGCGTGAT-3′; GAPDH-Reverse: 5′-GAGTGGGTGTCGCTGTTGA-3′. SYBR Green qPCR SuperMix for quantitative PCR was purchased from Vazyme Company. Quantitative PCR was performed using the ABI PRISMt-test, two-way ANOVA, or log-rank Cox was used for the statistical analyses. P <0.05 was considered statistically significant.We used GraphPad Prism version 8 (GraphPad software) for statistical analyses. Data were presented as mean ± standard deviation (SD). Student’s n = 222, P = 0.011, P = 0.015, P < 0.05, P < 0.05, HR > 1 in univariate analysis) such as clinical stage, tumor invasion, lymphatic invasion, distant metastasis, and QKI expression were taken into the multivariate mode, QKI expression, lymphatic invasion, and distant metastasis had predictive value for OS in patients with NSCLC . Overexpression of QKI-6 inhibited tumor cell proliferation, and knockdown of QKI-6 promoted cell proliferation . Furthermore, overexpression of QKI-6 significantly reduced cell colony formation ability  and induced a block in the G0/G1 phase. Conversely, knockdown of QKI-6 in A549 cells increased the proliferation and the proportion of cells in the S and G2 phases . Moreover, overexpression of QKI-6 significantly inhibited A549 cell migration, and knockdown of QKI-6 promoted A549 cell migration .To determine whether QKI-6 could exert anticancer effects, QKI-6 overexpression and knockdown were performed in A549 cells  database, which provided networks of molecular function  by Kaplan–Meier analysis. Higher AGR2 mRNA expression levels were correlated with poor prognosis . Thus, QKI-6 is likely an inhibitor of NSCLC metastasis and suppresses EMT and metastasis by regulating AGR2 via EGFR–SRC–STAT3 signaling.Among the differentially expressed proteins, AGR2 was significantly upregulated in sh-QKI-6 A549 cells compared with sh-NC. We represented the result of AGR2 OS in the TCGA sample . However, the low precision and inaccuracy of LFQ was a technical challenge. We tested the novel gene AGR2 involved in the positive regulation of the EGFR signaling pathway, which played an important role in the metastasis of NSCLC . AGR2 is a potential oncogenic biomarker, which is overexpressed in many patients with NSCLC with short survival duration  to identify genes that are differentially expressed in sh-QKI-6 and sh-NC. The LFQ proteomics approach improved our understanding and provided clearer insights into the QKI-6-mediated molecular changes and putative mechanisms and potential diagnostic biomarkers , 32. In duration –35. AGR2ignaling , 37. Howignaling . In the EGFR–SRC–STAT3 signaling plays a vital role in some human cancers. Dosch et al. reported that activation of EGFR–SRC–STAT3 signaling induced stromal remodeling and improved pancreatic cancer cell survival . Co-targTaken together, our results showed that QKI-6 could negatively regulate NSCLC cell proliferation, migration, and cell cycle progression. Furthermore, QKI-6 overexpression could downregulate AGR2 expression and inhibit the EMT process. Therefore, targeting this pathway could be a potential therapeutic strategy in patients with NSCLC.http://proteomecentral.proteomexchange.org/cgi/GetDataset, PXD027013.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The studies involving human participants were reviewed and approved by the Ethics Committee of Tangdu Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conceptualization: YN and TZ. Methodology: XS. Software: YN. Validation: HZ, JL, and XW. Formal analysis: LZ. Resources: DT. Data curation: LZ. Writing—original draft preparation: HZ. Writing—review and editing: FT and TZ. Visualization: YN. Supervision: FT and YN. Project administration and funding acquisition: YN. All authors have read and agreed to the published version of the manuscript.This work was supported by a grant from the National Natural Science Foundation of China (grant number 81301989 to YN).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.