Recognition that the loss of a loved one may result in prolonged grief disorder (PGD) has gained broad attention recently. PGD may disturb daily functioning to such a degree that mental health treatment is required. Because PGD symptoms often resemble symptoms of common mental disorders (CMD) such as anxiety, depressive, and post-traumatic stress disorder, clinicians may not consider a PGD diagnosis. Moreover, cultural varieties in expression of PGD may complicate recognition. This study explores the prevalence of PGD among both natives and refugees with anxiety, depressive, or trauma- and/or stressor-related disorders as well as clinicians' awareness and knowledge of PGD symptoms. In addition, a psychoeducation module on PGD symptoms is developed through patient expert collaboration.Prevalence of PGD symptoms is investigated among 50 participants who are referred to outpatient clinics for anxiety, depression, or post-traumatic stress, using the Traumatic Grief Inventory—Self Report Plus (TGI-SR+) and the Bereavement and Grief—Cultural Formulation Interview (BG-CFI). Clinicians will be interviewed on knowledge (gaps) with respect to PGD symptoms. Finally, focus groups with patient experts are held to develop a psychoeducation module tailored to the patients' needs, norms and values.This study will show prevalence of PGD among patients who are referred for anxiety, depression, and post-traumatic stress, awareness and knowledge of clinicians on PGD symptoms, and will offer patient expert informed psychoeducation.Research on prevalence and recognition of PGD is vital. Study results of the prevalence of PGD will be compared to previous studies. Recognition of PGD as distinct disorder from CMDs requires clinicians' awareness of symptoms related to the loss of a loved one. Thereby, clinicians need to take cultural aspects related to death, bereavement and mourning into consideration.The study protocol will be carried out in accordance with relevant guidelines and regulations. Exploratory research to assess the prevalence of PGD in patients suffering from CMDs will facilitate adequate diagnosis by increasing clinician's awareness of PGD symptoms. Tailored PGD psychoeducation, co-created by culturally diverse patient experts and clinicians will be made publicly available. Not long after we moved, the Arabic Spring started in Egypt and we were no longer safe. We then moved to Lebanon to start a new life free from violence. Unfortunately, a few days after my son got married, he died in a car accident in Lebanon. His passing turned the lives of my family upside down. We were not able to accept the loss of our son and we were sad all the time. At the same time the war situation in Syria became worse. The company that I had built up from the bottom got bombed. We then decided to flee to the Netherlands. In the Netherlands, I got the diagnosis of a post-traumatic stress disorder and a depression. Four years of treatment according to the protocols followed, by many different clinicians. I received medication and trauma treatment, but without any result. When I entered the sixth treatment with yet another clinician in a different mental health institution, he started paying attention to the loss of my son. At last I had the feeling that the core cause of my mental health problems was being addressed. The treatment of the grief over the sudden and unexpected loss of my son was confronting, but healing.”This reflection of a Syrian refugee patient who first started to experience relief of his mental health problems after discussing the loss of his beloved son does not stand alone.In the past 20 years, recognition that the loss of a loved one may influence psychosocial wellbeing has increased, especially if that loss is unexpected or the result of a traumatic/overwhelmingly negative experience. Following the loss of a loved one, practical, organizational, financial, and sometimes juridical issues dominate the first period. When these issues have been managed, processing the loss may start. For most people supportive contact with one or more family members, a friend, a close acquaintance, a peer, or a pastor/priest/imam/rabbi or any other religious authority, is sufficient for processing the loss. However, for some who have experienced the loss of a loved one the grief process does not get started, stagnates, or results in serious mental health problems that might require mental health care. They may develop Prolonged Grief Disorder (PGD), a disorder included in the text revision of the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5-TR)  and the Diagnostic criteria for PGD according to DSM-5-TR  are summMeta-analytic studies indicate a pooled prevalence of 9.8% for PGD among non-psychiatric, adult populations exposed to non-violent bereavement , and a pProcessing grief following bereavement involved meaning attribution to loss, a process that is determined by individual, social and cultural factors as well as factors related to the circumstances of the death and the relationship with the deceased . ExampleAnother research gap this study seeks to address is the awareness of PGD as a unique symptom profile among clinicians. In the diagnostic process, the focus of clinicians is often on PTSD and depression and symptoms are not always related to the loss of a loved one. Especially in the case of refugees, PGD may be overlooked, despite a high prevalence among this group . AwareneAwareness of cultural differences is also essential when providing patients with psychoeducation as part of their treatment. Adequate psychoeducation is necessary to improve patients' understanding of their symptoms and enhance effective treatment. Consulting patient experts when creating psychoeducation increases the likelihood that psychoeducation will match the patients' needs. To our knowledge, no psychoeducation on PGD has been developed with the input of culturally diverse patient experts.The present study aims to (1) assess the prevalence of PGD in a native and refugee patient population referred for treatment of anxiety, depressive or trauma and stressor-related disorders, and, in addition, to explore differences in cultural aspects of bereavement and grief between these groups, (2) to explore awareness and knowledge of PGD among clinicians at secondary outpatient clinics of Drenthe Mental Health Care, and (3) to develop patient expert assisted psychoeducation of PGD tailored to the patients' needs.This study has a mixed methods design that contains qualitative as well as quantitative methods. First, presence of symptoms of PGD and CMDs in participants will be quantitatively measured. Participants with clinical and subclinical scores will be interviewed on bereavement and grief for in-depth insights into the participants' perspective on loss, bereavement and grief symptoms. Interviews with clinicians will focus on the recognition of mental health symptoms consistent with PGD. Focus groups with clinicians and patient experts on the diagnostic and treatment of PGD will be organized to develop person-centered and culturally sensitive psychoeducation. Clinicians will be trained in the newly developed psychoeducation. After they have delivered psychoeducation to their clients, debriefment for feasibility, acceptability, and clinical utility fill follow. The procedure of the study is represented in During the timeframe of this study, patients who are referred to either a mental health unit for transcultural psychiatry in two cities (Beilen and Heerenveen) or outpatient clinics for CMDs in the city of Assen will be screened by professionals involved in the triage and/or intake on having lost (a) loved one(s) and other inclusion and exclusion criteria . The recAfter consent, 10 mental health care professionals will be asked to participate in a semi-structured interview on their knowledge, view and skills concerning PGD. The interview consists of eleven open-ended questions concerning the mental health problems of their patients, knowledge about PGD, experience with patients who have lost a loved one, experience with bereavement and grief in clinical practice, and sensitivity toward symptoms related to bereavement and grief.via Zoom and will be recorded after consent from participants.During this study, 10 focus group meetings will be arranged with both native and refugee patient experts and mental health care professionals in order to discuss relevant topics concerning PGD, including norms and values with respect to bereavement and grief. All patient experts and mental health care professionals will receive information on the study and will then be asked if they are willing to participate. The focus group meetings will take place online The focus groups will contain in total of six up to 10 mental health care professionals, patient representatives, and patient experts. Patient experts with and without a migratory background will be recruited from formal patient expert education. Patient representatives are refugees with experience in mental health research who have not been a patient themselves. The inclusion of patient experts aims to focus on the patients' needs, norms and values from their own experiences.Fifty patients are needed to include for the descriptive statistical analysis. We aim to include 25 participants in the refugee and 25 participants in the native group. Based on prevalence numbers of PGD in several studies, we aim to include fifty patients to meet the required number of twenty respondents who score above threshold levels for PGD according to DSM-5-TR and ICD-11 on the TGI-CA. The number of twenty patients is estimated as feasible, while valid statements can still be made on the basis of the results. Only descriptive and exploratory comparative statistical analyses will be carried out. Participants will be recruited from outpatient clinics patients within Drenthe Mental Health Care, department of transcultural psychiatry and the outpatient clinics for CMDs. We deliberately include both native and refugee patients in equal proportions in this research in order to establish a diverse study population. A culturally diverse study population enables generalization to a variety of patients when it comes to recognizing PGD and providing psychoeducation. In addition, a culturally diverse study population enables us to make a comparison between native and refugee patients. Inclusion and exclusion criteria are listed in To assess PGD, the Traumatic Grief Inventory—Clinician Administered (TGI-CA) will be employed among participants . The TGITo assess the prevalence of psychological symptoms as listed in the DSM-5, the Miniscan will be applied. The Miniscan is a shorter version of the Schedules for Clinical Assessment in Neuropsychiatry (SCAN) of the World Health Organization . It's a The Bereavement and Grief Cultural Formulation Interview (BG-CFI) will be used to explore the context and the perspective of patients on loss, bereavement and grief symptoms. The interview will be conducted by researchers with participants who have a (sub)clinical score on the TGI-CA. The interview is available in English and Dutch , 20. If The development of psychoeducation about PGD will be facilitated in 10 focus group meetings with mental health care professionals and patient experts. The meetings of this focus group will take place online. Each meeting will have one central theme. Themes that will be discussed are, for example: what should we be aware of when talking about grief? What can be discussed and what can not? What to do when a patient is hesitant to talk about grief? Attention will be paid to adapting psychoeducation to the knowledge level of patients, using language that is adapted to patient's understanding, being aware of communication barriers and subsequently discussing and solving them, and to a connection between a theoretical orientations and practical applications.The BG-CFI and patient expert informed psychoeducation will be evaluated through an adapted version of the Debriefing Instrument for Patients (DIP). This instrument has previously been used in research on the Cultural Formulation Interview, which is included in the DSM-5 . The DIPReports of the focus group meetings, the BG-CFIs and open-ended questions of the DIP and DIC will be qualitatively analyzed on most relevant themes that need to be addressed in the development of psychoeducation. Computer-assisted qualitative data analysis software Atlas.ti 8.0 will be used for in-depth qualitative thematic analysis . Atlas.tThemes of the focus group meetings will concern experiences with grief and bereavement; communication between patient and clinician; required information; barriers for reprocessing; the role of family, loved ones and others; and attitude of clinicians during treatment. Retrieved information will be thematically analyzed and reviewed in the focus group by means of member check to raise credibility . PatientResults from the Miniscan and the TGI-CA will be used to answer the research question that concerns the prevalence of PGD according to DSM-5-TR and ICD-11 in the research population and comorbid diagnoses. Descriptive analysis will focus on comorbidity of PGD with PTSD, depression, and generalized anxiety disorder. Exploratory comparative group analyses will focus on prevalence of PGD according to DSM-5-TR and ICD-11 and comorbidity of PGD with PTSD, depression, and generalized anxiety disorder.Recent developments in the classification of mental disorders have resulted in the recognition of symptoms that are related to PGD. Recognition of PGD in patients who suffer from CMDs is both relevant and complicated because of symptom overlap between PGD, PTSD, and depression. Clinicians may classify CMDs with neglect of the potential relation of these disorders with the loss of a patient's loved one. In clinical practice, distinctions between symptoms related to PTSD or depression may not be clear to the clinician if the question whether there is loss of a loved one is left unasked. The clinical case example in the introduction shows that failed or late recognition of PGD may result in inadequate treatment and unsuccessful response.Although cultural variations in response to bereavement and grief such as cultural traditions related to death, bereavement, and mourning may be omnipresent, cultural aspects of PGD are under researched. To facilitate clinical exploration of those cultural aspects, a Bereavement and Grief Cultural Formulation Interview (BG-CFI) has been proposed . FeasibiStrengths of this study are that both CMDs and PGD will be assessed allowing diffusion in diagnosis to be elicited, that cultural variations related to death, bereavement, and mourning will be included, and the involvement of (native and refugee) patient experts in the development of psychoeducation. Limitations of the study are the potential influence of implementing the study on the awareness of PGD among clinicians, and stigma-related, attitudinal, or instrumental barriers to participate in the study.The study protocol has been submitted for ethical approval at the University Medical Center Groningen . The Medical-Ethical Review Commission decided that ethical reconsideration was not required as a clinical study. However, the study protocol was then submitted for ethical approval of the Psychology Department, University of Groningen (PSY-2021-5-0527) and approved. Exploratory research to assess the prevalence of PGD in patients suffering from CMDs is necessary, because of the potential risk that clinicians tend to classify presented symptoms without consideration of the relation with the loss of a loved one. A lack of such consideration may lead to classification of anxiety, depressive, or trauma and stressor-related disorders, while symptoms may be related to PGD. PGD requires different types of treatment, or a focus in treatment on the relation between the symptoms and bereavement and grief. The case study example clearly shows that four types of trauma-focused treatment did not result in symptom reduction, whereas treatment focused on grief and bereavement did. Therefore, the main ethical aim of this study is that patients suffering from PGD obtain accurate diagnosis and treatment.Another ethical aspect of this study is the inclusion of patient experts in the development of psychoeducation on PGD. Often psychoeducation has been developed top-down without patient consultation. Such development may result in ineffective psychoeducation that insufficiently connects to the patient's needs. Therefore, focus group methods are applied in this study including patient experts to represent patient perspectives in the development of psychoeducation on PGD. Moreover, due to increasing cultural diversity of patient populations, the inclusion of refugee patients, and to engage into present-day diversity and inclusion requirements, this study focusses on patient's norms and values as well. Therefore, the selection procedure for members of the focus group will include cultural diversity preferences. Patient-informed focus group meetings will be applied to gather most important themes of PGD, the patient's needs, and norms and values related to grief and bereavement. Gathered information will be analyzed using computer-aided qualitative analysis. The analysis will be peer debriefed in focus group meetings and applied in psychoeducation folders. The study results will be presented at relevant national and international symposia.SG is the primary investigator and has drafted the manuscript. MM, DC, and GS have revised and approved the final version of the manuscript. All authors contributed to the article and approved the submitted version.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.


Maintaining a high level of animal welfare is essential in zoos, sanctuaries and aquaria for ethical, legislative and functional reasons. Therefore, it is necessary to have welfare assessment protocols that can be incorporated into daily management programs. Currently, there are different approaches to assessing animal welfare in zoos. Those that can be applied to multiple species consist of checklists or qualitative assessments, with limitations, especially regarding the lack of guidance in the selection and interpretation of indicators. Validated protocols also exist, but they are for very few wild species. This study aimed to develop, test in the field, and describe an animal welfare assessment protocol for wild animals under human care, that can be applied to multiple species, intended to overcome the use of generic welfare checklists and offer an alternative to challenging and time consuming species-specific tools.The development process consisted of the elaboration of a protocol, substantiated by published literature on zoo animal welfare and multidisciplinary focus group work, and its on-field feasibility test. This was performed on 14 species of different taxa housed in an Argentinian zoo. The protocol was structured in two forms: an initial form to serve as scan using various animal-based (ABM), resource-based (RBM), and management-based measurements (MBM), and a follow-up form using exclusively ABM. The protocol also included a user's manual with information about preliminary preparation, equipment required, steps from arrival until completion, and details on how to assess each indicator. The scoring method consisted in rating each indicator on a 3-point scale.23 ABM, 19 RBM, and three MBM were tested and selected to integrate Ackonc-AWA, a multidimensional protocol covering the five animal welfare domains and applicable to multiple species.This protocol was entirely developed in Spanish and can be applied noninvasively and at a low cost, which constitute features of high relevance for Latin America. Further applications of the described welfare assessment tool in other species and different institutional contexts will reinforce the validation of the proposed measurements and allow the systematic and routine evaluation of animal welfare in zoos. Individual animal welfare and species welfare are critical obligations of zoos, sanctuaries, and aquaria (hereafter simplified as “zoo(s)”). Even the most ambitious conservation goals will not be adequate justification for keeping wild animals in captivity if zoos do not actively demonstrate high standards of animal welfare –4. The iThe past few decades have seen an increased interest in animal welfare among researchers and zoo staff. Zookeepers identify training in this area as relevant and important to their work  and the According to the Single Public Registry of Wildlife Operators , in ArgeEnsuring animal welfare requires knowledge, experience, and institutional commitment, as well as the deployment of comprehensive and robust animal welfare assessment tools, which can be implemented at two levels: institutional  or individual  , 16. As The approach in the construction of protocols to assess animal welfare, their methods, and the way in which they should be evaluated or validated depend on the goals, which need to be clearly defined before starting the development process. Botreau et al.  identifyCurrently, there are different tools to assess animal welfare in zoos. Those that can be applied to multiple species usually consist of extensive checklists with questions aimed at revealing what the conditions of the physical and social environment are like and provide insight into the welfare of an individual animal e.g., , 16, 24], 2416, 2This study aimed to develop, test in the field, and describe an animal welfare assessment protocol for wild animals under human care, which can be applied on a daily basis, noninvasively, and at a low cost, under the aforementioned prescriptive model. That is, first the current welfare status of the animal is assessed to understand the starting point and then its evolution is monitored by collecting information that allows the development of tailor-made recommendations and rapid decision making. Hence, it was intended that the protocol would be able to provide two types of assessment: comprehensive  and regular (frequent monitoring to detect early deviations). Simultaneously, it aimed to obtain an intermediate solution between protocols that are easy to apply yet rely entirely on the judgment of the assessors, and validated but species-specific protocols that are useful only for assessing the species for which it was developed.The protocol was tested at an Argentinean zoo, member of Asociación Latinoamericana de Parques Zoológicos y Acuarios (ALPZA) and World Association of Zoos and Aquariums (WAZA), which was in the process of transformation and restructuring. The protocol was applied and tested between October and December 2017.The protocol was given the name Ackonc-AWA, which combines the purpose of conducting animal welfare assessments (AWA) with the role of the individuals involved in the observation and data collection process , given that the phonetics of the name reflects the native Andean word “ackoncahua” which is translated as “sentinel”.The conceptual animal welfare framework adopted to create Ackonc-AWA protocol was the Five Domains Model , with a The principle of feasibility was taken into account for the selection of the welfare indicators , 33. TheIn addition, two meetings were held with eleven representatives of different areas of the zoo  to submit their input to a multi-disciplinary discussion in a focus group in order to select agreed upon items for assessment, as a way to provide content validity .Once the first selection of the indicators to be assessed had been made, two types of forms were developed: an initial form and a follow-up form. The initial form consisted on 45 indicators  , which wAdditionally, a user's manual was written with instructions on the method used to assess and score each indicator. Both the indicators selected and the instructions for their assessment were the same for the different species and individuals included in the pilot test, although changes and clarifications were made in the user's manual to adapt them to the differential characteristics of each taxon.Before beginning the assessment, sentinels had to be familiar with the following information about the species to be assessed: biological and behavioral features (including specie's ethogram); housing and handling requirements recommended by international associations; nutritional information  and both routine and scheduled activities . Likewise, sentinels should have a layout/map and information about the location and dimensions of the enclosure.Every effort should be made to minimize the impact of the presence of the sentinels on the behavior of the animal under study. Sentinels should remain out of sight and avoid any kind of interaction with the observed animal during the data collection to minimize the impact of his or her presence on the behavior of the animal under study .Indicators were rated on a 3-point scale . For indicators that could be rated in several contexts , rating was made according to the context that represented a higher level of animal welfare compromise. When any indicator was rated “B” or “C”, the sentinel provided additional information about this on the “Notes” column.Hydrochoerus hydrochaeris), to explore the potential usefulness of the protocol for group assessment, with proper modifications or adaptations.The selection of the species and individuals on which the protocol was tested was based on the following inclusion criteria: 1—prospective permanence of the animals in the zoo: longer than 2 years; 2—easy identification: phenotypic characteristics or features that made it possible to individualize the animals housed in groups; and 3—include species from different taxonomic categories to test the ability of Ackonc-AWA protocol to be applied for different taxa. As a result, 14 individuals  from different orders and families were selected . Ackonc-Sentinels assigned to observe, record and score the indicators on site were selected from the different areas involved in animal management and care, based on interviews, in search of those who met a combination of experience, training, predisposition and observation skills. Their election was also agreed with representatives of the institution in order to avoid hindering or disrupting daily activities. Hence, the team of sentinels consisted of a group of three external veterinarians experienced in animal welfare assessments  and a group of nine zoo staff members with no prior experience in animal welfare assessments, belonging to different departments .All inexperienced sentinels received a 4 h theoretical and practical training on animal welfare assessment in general and on the use of the protocol in particular, designed and delivered by the researchers. A virtual library was also created with ethograms and information on each of the 14 species' nutritional, physiological, environmental and behavioral needs, selected by the researchers from books, husbandry manuals and peer-reviewed scientific publications. All sentinels were given access to this virtual library and were instructed to read the documents selected for the corresponding species before beginning the on-field feasibility test.The researchers and the zoo staff from the AWMP Department were exclusively dedicated to this task, so they evaluated the entire protocol . On the other hand, the rest of the sentinels were assigned a different number of indicators to score, since they had different availability to collaborate with this research . For the®  or under specific situations (courtship behavior outside the reproductive season). In such cases, sentinels were instructed to use the legend “does not apply” to differentiate them from those that could be left blank due to other reasons .The average completeness of the forms was determined by averaging the degree of completeness achieved by all sentinels for all species. In addition, a ranking of the indicators most often left blank was made by counting the number of times that each indicator was not evaluated when it should have, in relation to the total of forms  across species and sentinel groups.Ackonc-AWA protocol was designed so that the observations and completion of the forms can be done by the zookeepers, combining this activity with their other responsibilities. Therefore, it was important to determine the degree of difficulty perceived by the staff in applying the chosen indicators. For this purpose, after completing the Ackonc-AWA forms, each sentinel was asked to assign a degree of difficulty to fill out each form between 1 and 10, with 1 being the minimum and 10 the maximum. At the bottom of each form, the sentinels had to specify which indicator was found as the most difficult to evaluate. With these responses, the indicators were rated for their level of difficulty, from the most often reported to the least often reported. The results were analyzed by averaging the degree of difficulty assigned for all species and sentinels, differentiating between initial and follow-up assessment forms.It was intended that the Ackonc-AWA protocol require <2 h per individual or group for data collection since long application protocols have more difficulties to be used regularly in zoological institutions, especially in those lacking resources or exclusive personnel for this purpose, a very frequent situation in Latin America. The average time (in minutes) required to complete the two welfare assessment protocol forms was recorded for all species and sentinels, differentiating between initial and follow-up assessment forms.Ethical review of the project was requested to the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the Faculty of Veterinary Sciences of the University of Buenos Aires, and a review exemption was granted given the observational nature of the project. The study focused on the non-invasive/intrusive assessment of animal welfare, so no interventions of any kind were carried out on the animals. There were no potential adverse effects, nor foreseeable risks or hazards associated with this project, with regards to animal, plant and/or human wellbeing. The participation of zoo staff in this study was completely voluntary and under written informed consent. The survey responses were strictly confidential and data from this research was reported only in the aggregate. The information was coded and remains confidential.The mean intra-observer proportion of agreement was 0.8652 among the sentinels and 0.8678 among indicators . The mean observed Kappa was 0.7763 among the sentinels and 0.7681 among indicators, which on the Landis and Koch  scale isThe average completeness for the initial form was 86.21% whereas for the follow-up form it was 79.07%. The top ten indicators most often left blank were part of both assessment forms and were therefore analyzed together. No indicators were left blank over 50% of the times. Only Water intake was left blank over 40% of the times . Three indicators were left blank between 40 and 30% of times: Micturition behavior , Defecation behavior , and Use of environmental enrichment ; two indicators were left blank between 30 and 20% of times: Social behavior  and Reproductive behavior ; and four indicators were left blank between 20 and 10% of times: Hooves/claws/teeth , Agonistic behavior , Food intake  and Behavioral diversity . The rest of the indicators included in the protocol were left blank <10% of the time.The mean reported difficulty across species and sentinels was 4.79 +/– 1.13 for the initial form and 5.20 +/– 1.51 for the follow-up form. Analysis of sentinel responses showed that the indicator most frequently reported as difficult to assess was Behavioral diversity  followed by Defecation behavior , Micturition behavior , Hooves/claws/teeth , Water intake  and Food intake .The average time across species and sentinels required to complete the initial form was 51.32 min. +/– 29.36 min. Completion of the follow-up form took an average of 58.32 min +/– 23.11 min. In the face of on-field feasibility results, some changes were implemented for the assessment of the indicator “Behavioral diversity” within the Behavioral and mental domain by the introduction of activity budget sheets of 20 min each, on three  different time slots see . After sNo changes were made for the indicators included in the Nutritional, Environmental and Health domains. As a result, a total of 45 indicators  were selected to integrate Ackonc-AWA, covering the five animal welfare domains.Three ABM  and seven RBM  were selected to assess the nutritional domain. Twelve RBM  and the three MBM  were adopted. Ten ABM  were selected to assess the health domain. Given the multispecies purpose of the Ackonc-AWA protocol, it is important to note that for some species  it may be necessary to score the indicators “Defecation behavior” and “Stool score”, together with “Micturition behavior” and “Urine appearance” respectively, due to their physiologic and anatomic features.Ten ABM  were selected to assess the Behavioral and mental domains. This study introduced an innovative multi-species animal welfare assessment protocol for wild animals under human care, intended to overcome the use of generic welfare checklists and offer an alternative to challenging and time consuming species-specific tools . Ackonc-Therefore, in order to successfully implement the current protocol, prior preparation is a key stage when used on a species for the first time. This includes reviewing the most updated guidelines for the adequate maintenance of the species in captivity and its dietary, health, environmental, behavioral, and affective needs , 38. SomThe need for prior search for information on the species and, if not available, the realization of an ethogram, could take considerable time. This time may be longer or shorter depending on the species, since for some there are husbandry manuals and abundant bibliography, and for others information is very scarce or absent. This prior preparation could be seen as a limitation in comparison to other tools. However, it is important to note that this procedure is done only once at the beginning of the assessment and then the sentinels use the protocol adapted to the species of interest, without the need to go back to the literature for each assessment. In the field trials, the average time used by the sentinels was 51.32 min +/– 29.36 min for the initial form and 58.32 min +/– 23.11 min for the follow-up form. Even with the addition of activity budgets , an increase in the time required for assessment is not expected, as many indicators can be assessed during the same observation. Nevertheless, this should be evaluated in further studies.Ackonc-AWA implementation cost is low, it is non-invasive/intrusive and takes relatively little time. Although these are all desirable qualities for any animal welfare assessment protocol , they coIt should be noted that the Ackonc-AWA protocol includes some indicators that can be assessed by close observation and even palpation . This is so that future users of the protocol are able to collect the information in the most practical way for them, as many ABM can be assessed by training and conditioning or during a scheduled veterinary capture. Given that zoos frequently train animals to cooperate in veterinary maneuvers without the need for physical or chemical restraint, and that there is abundant scientific evidence indicating that operant conditioning training is another strategy to improve animal welfare in zoos and the human-animal bond, and is even a form of environmental enrichment –42, closIn order to further increase its practicality, Ackonc-AWA was designed in two forms: initial and follow-up. Although, as discussed above, preparation requires some time, once the protocol has been adjusted to the species under study, the follow-up form can be applied as often as necessary, even on a daily basis. Its practicality and low cost of implementation is partly based on the fact that, subject to prior training, it can be applied by the institution's own personnel and done in the context of their daily duties.In this regard, a core component of developing and using animal welfare assessment tools in zoos is to leverage the experience and expertise of the staff , 43, 44.Simplicity of implementation is also a key factor for the feasibility of animal welfare assessment protocols. Although the overall feasibility results were positive, adequate training and coaching could be implemented to reduce some of the difficulties encountered by sentinels when filling out the forms in animal welfare assessments. As demonstrated by Rodríguez Ruiz and Heredia Rico , traininThe indicators most frequently reported as difficult to assess were “Behavioral diversity”, “Defecation behavior” and “Micturition behavior”. In order to simplify the assessment of “Behavioral diversity”, the use of activity budget sheets through focal  and scan (group) sampling was incorporated, as it is an objective, quantitative and validated method for animal welfare assessment in zoos , 51, 52,The need for an analyst can also be discussed as a possible disadvantage. Nevertheless, the analysis and interpretation of the information obtained from behavioral budgets has been widely used in zoos, and many of the institutions in Latin America have highly trained personnel within their staff to perform this task.To assess affective states, Ackonc-AWA proposes a joint approach of the behavioral domain with the mental domain. This is because some affective states are directly or indirectly assessed in this protocol using behavioral indicators. Due to the type of institutions for which this protocol was designed, the importance of assessing affective states in relation to the human-animal bond is emphasized. The effects of the visitors and zookeepers over the animals' experiences and their consequent welfare state are addressed in the protocol through two ABM indicators: Reaction to strangers and Interaction with zookeepers. As stated by Mellor et al. , Domain All of these responses tend to offer an approach to affective states in relation with the interactions that animals and humans have. In the future, further interventions on negative or positive human attributes and attitudes toward animals could be useful to address this issue from another perspective, in order to acquire a MBM that could operate as a welfare predictor.The qualitative nature of this protocol may be considered controversial. Observer ratings are scores given to a variable using units of measurement defined by the researchers. Since they involve subjective judgments, some researchers question whether they can be trusted to reflect reality in an unbiased manner . HoweverWith regards to the final assessment results, Ackonc-AWA provides a representation of an animal's welfare and a temporal component that is easy to read and allows tracking changes over time, making it possible to differentiate between problems that affect animal welfare at the current time and those that pose a risk to animal welfare in the medium and long term. As a protocol with a prescriptive approach, it does not give a final numerical result, but looks at each indicator in order to identify potential welfare concerns, which prevents the institution from settling for an acceptable overall result that could be deceiving and could pose a severe threat to animal welfare. For example, a zoo that scores 8 out of 10 might be satisfied with the idea that it has a good overall score and not work on establishing a plan to improve those indicators that were found to be compromised. The situation of these compromised indicators could become chronic and begin to impact negatively on others that were adequate. On the other hand, by letter-marking indicators it is easy to identify those that require immediate resolution and establish a prioritization plan. The proposed 3-point scale score would facilitate a fast and practical prioritization of the identified welfare concerns, and to tag the more urgent correction actions.ex situ conservation efforts do not harm (or do as little as possible) the welfare of individuals  was tested on 14 species of different taxa housed in a zoo in Argentina. Representatives from different areas of the institution as well as 3 of the authors participated in the test. It was possible to demonstrate the feasibility and test-retest reliability of the protocol. However, due to time limitations of the institution staff, its inter-observer reliability has yet to be tested.As a result of this process, Ackonc-AWA, a multidimensional protocol for welfare assessment in multiple animal species under human care, was obtained. This proposal offers an intermediate solution between protocols that are easy to apply yet rely entirely on the judgment of the assessors, and validated but species-specific protocols that are useful only for assessing the species for which they were developed.Further applications of the described welfare assessment tool in other species and different institutional contexts will reinforce the validation of the proposed measurements and allow the systematic and routine evaluation of animal welfare in zoos.The original contributions presented in the study are included in the article/Ethical review of the project was requested to the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL) of the Faculty of Veterinary Sciences of the University of Buenos Aires, and a review exemption was granted given the observational nature of the project. The study focused on the non-invasive/intrusive assessment of animal welfare, so no interventions of any kind were carried out on the animals. There were no potential adverse effects, nor foreseeable risks or hazards associated with this project, with regards to animal, plant and/or human wellbeing. The participation of zoo staff in this study was completely voluntary and written informed consent was obtained from all participants. The survey responses were strictly confidential and data from this research was reported only in the aggregate. The information was coded and remains confidential.DR, AF, and LR contributed to the elaboration of the first drafts of the protocol, interviewed the zoo staff, met with the zoo staff/focus group to test validity, designed the on-field test, trained the staff as sentinels, organized the scheduled observations for all the sentinels, did field work as sentinels, analyzed the results of the observations including feasibility results, and elaborated the Ackonc-AWA protocol. DR contacted and coordinated actions with the institution where the pilot test was conducted and wrote and edited the first draft of this manuscript. LR and AF wrote sections of the manuscript and edited the first draft and manuscript. CB was responsible for the statistical analysis of the results for test-retest reliability and edited the manuscript. OT-P collaborated as the main counselor for methodological aspects of the study and edited the manuscript. All authors contributed to manuscript revision, and read and approved the submitted version.


One of the biggest economic and environmental sustainability problems is the over-reliance on petroleum chemicals in polymer production. This paper presents an overview of the current state of knowledge on biopolymers combined with biosensors in terms of properties, compounding methods and applications, with a focus on medical and environmental aspects. Therefore, this article is devoted to environmentally friendly polymer materials. The paper presents an overview of the current state of knowledge on biopolymers combined with biosensors in terms of properties, compounding methods and applications, with a special focus on medical and environmental aspects. The paper presents the current state of knowledge, as well as prospects. The article shows that biopolymers made from renewable raw materials are of great interest in various fields of science and industry. These materials not only replace existing polymers in many applications, but also provide new combinations of properties for new applications. Composite materials based on biopolymers are considered superior to traditional non-biodegradable materials due to their ability to degrade when exposed to environmental factors. The paper highlights the combination of polymers with nanomaterials which allows the preparation of chemical sensors, thus enabling their use in environmental or medical applications due to their biocompatibility and sensitivity. This review focuses on analyzing the state of research in the field of biopolymer-sensor composites. Biosensors are diagnostic tools used in many areas of life: medicine, the environment, the packaging industry and many others ,2,3,4,5.Modern sensors are characterized by high sensitivity, selectivity, on-time response, stability, reversibility, are low-cost, and are easy to use. Their great advantage is the quick reaction to the presence of the monitored factor, which allows for quick intervention.The development of chemistry and materials engineering creates enormous opportunities to develop new solutions, the application of which in everyday life is a milestone in health protection, the environment, food waste, agriculture and many other areas .Chitosan, alginate, cellulose and starch are some of the natural polymers that are widely used in conjunction with sensors. The wide range of applications of biopolymers results from their properties, such as their capability to collect and accumulate analytes on sensor surfaces, and the possibility of producing and using various forms . The scientific interest in the use of biosensors in combination with biopolymers in many fields of science is growing every year. This paper presents an overview of the current knowledge on biopolymers in combination with biosensors in terms of properties, methods of linking and applications, with particular attention to the medical and environmental aspects. The current state of knowledge as well as future perspectives were presented. Polymers play an important role both in the biological world and in its present industry. Many natural polymers, for example proteins and nucleic acids, transfer fundamental biological data, whereas other polymers such as polysaccharides contribute fuel for cell functions and serve as constructive elements in living structures . BiopolyBiopolymers are materials that have potential applications in all sectors of the economy. Polymer composites are biocomposites if at least one constituent is biobased or biodegradable. The terms biodegradable polymer and biopolymer are often used interchangeably in the literature, but there is a major difference between the two types of polymers  that und- The source of the raw materials; - The biodegradability of the polymer.Considering the two criteria for the division of polymer compounds, i.e., the source of origin of raw materials and their biodegradability, the compounds shown in A different approach regarding the division of biopolymers is according to monomeric units. It is possible to differentiate three different groups  10,11].,11.10,11When the monomeric unit consists of polysaccharides, there are carbohydrate chains, which can be branched or linear joined by glycosidic linkage. Examples of such monomeric units are starch and cellulose. Protein units are made up of amino acids which can be represented by collagen or fibrin which are linked with peptide bonds (amide linkage). The third group, which is a protein monomeric unit, consists of nucleic acids which can be differently arranged in long polymer chains made of at least 13 units. Examples are DNA and RNA . Biopolymers can be produced both by biological organisms, e.g., plants and animals, and chemically synthesized from natural starting materials, e.g., corn, starch, etc. A detailed classification of the biopolymers was discussed by Dziuba et al. . BiopolyComposites are materials that comprehend two or more chemically and physically diverse phases isolated by a separate interface. They are a mixture of materials differing in composition, where the particular components maintain their unique identity. Summary composite materials are a combination of two or more components, one of which is present in the matrix phase and the other may be in the form of particles or fibers. CMCs have high creep resistance and thermal shock resistance. CMCs are fabricated by hot pressing, hot isotactic pressing and liquid phase sintering techniques and are used as cutting tool inserts for machining hard metal alloys with a tool life greater than cemented carbides. Ceramic composites improve the fracture durability of material ,16. PMCsAdvanced composites of fibers and grid combinations yield superior quality, mechanical strength, thermal stability, hardness and stiffness ,20. GlasThese differing elements act together to produce the essential mechanical strength of the composite part . A speciThe advantage of composite materials is the ability to design a range of properties that may vary depending on the intended use of the composite. The key advantage of these materials is their programmability, which now allows them to be widely used throughout the world around us and in various areas of life, such as construction, industry, medicine, production of everyday objects, and even devices used in space. Today, biocomposites and biopolymers with controllable lifetimes have become major topics for different areas of applications. This article focuses on the use of biocomposites along with sensors for environmental and medical applications.A key question to be answered is what a biosensor is. This is a tool that can be used for obtaining the physical identity, which is then translated into a recognizable signal. It should have the ability to react with different biological components to react in the desired way. The biosensor can detect for example biomolecules such as urea, estrogen, glucose, or cholesterol . Sensitivity that is for example the concentration of the influencing element;Selectivity, so the sensor will not react to every molecule but just the expected one;The range is strictly connected with sensitivity and corresponds to the concentration range where the sensitivity is active;Response time, how much time is required for a biosensor to indicate 63% of the final response;Reproducibility, the accuracy of the sensor’s output,Detection limits its lowest detectable concentration;Lifetime is the period when the biosensor can be used without significant loss in performance;Stability, if within a fixed period there is any change in the baseline or sensitivity .Eight major parameters characterize the biosensor:The classification of biosensors can be based on the mode of physiochemical transduction or the type of biorecognition element  38,39,4,439,40.Electrochemical biosensors are the most widely described and investigated due to their properties. They can be characterized by a low detection limit, simplicity of construction, specificity and ease of operation . The operation of optical biosensors is based on the occurrence of a biochemical reaction, which is connected with light absorption or emission. Absorption, luminescence, fluorescence, surface plasma resonance, etc., are among the techniques linked to this group of sensors. The visible color change makes colorimetric sensors the most explored . Because most of the biochemical reactions involve a change in enthalpy and heat changes, it is reasonable to use the phenomenon in biosensor design . Piezoelectric biosensors use piezoelectric crystals that participate in the measurement of the mass change by correlating with the change in oscillation frequency. This phenomenon is a result of biomolecular interaction . A special class of biosensors is conducting polymers (CPs), which contain large resonation structures with many sp2- carbon atoms that enable the delocalized transport of charge carriers. Synthetic metals such as polyaniline (PANI), polypyrrole (PPy), polythiophene and derivatives are among the group ,44. Biosensors can also be classified based on the utilized transduces. Here, three generations can be distinguished. The first one includes biosensors, which use electrical responses that occur through the diffusion of reaction products to the transducers. A high-applied potential and fluctuant concentrations of the product, which can result in interferences, decrease in electrical currents and limits of detection, are among the main limitations of this group. The separation of the second generation was directly related to the application of mediators between the reaction products and transducers. The lack of selectivity of the mediator results in various interfering interactions. The third generation of biosensors revolves around the biochemistry and electrochemistry that occur as a semiconductor. This category is characterized by high selectivity and sensitivity ,46.Biosensors can be used in various applications shown in Bioresponsiveness is a property that allows the polymer to respond selectively to given molecules. An example of such behavior is chondroitin sulphate, which is a glycosaminoglycan and can bind to CD44 receptors which are commonly expressed in cancer cells. This can be used in recognition of the kind of cells as well as the attachment. What is more interesting is that the biopolymer undergoes degradation when in the environment where there is the presence of the enzyme lysosomal hyaluronidase, which is also commonly present in cancer cells. The ability to attach and degrade may be used in the targeted treatment of cancer cells by delivery of a drug. Similar behavior can be used for the diagnosis of wound infections. For this purpose, alginate or agarose matrix was used and peptidoglycan was covalently bound with Remazol, which is a blue dye. Primarily the matrix is yellow, but in the presence of lysosome and elastase in wound fluid hydrolysis occurs, releasing dye which changes color from yellow to blue. Next, interesting research was conducted by Wang et al.  and it wT of ATP aptamer caps from the surface of gold nanocage, in this way causing the opening of pores and releasing enclosed dye inside, causing a visible change of color. The important thing is that the treatment has high selectivity as it does not respond to ATP derivatives such as GTP, UTP, or CTP. The ability can be used for detecting cellular ATP in Ramos cells or in situ living Ramos cells. Schneider et al.  conducteA biosensor devoted to the sensing of glucose was also tested. It is based on immobilizing glucose oxidase into a polymeric carrier. This enzyme converts glucose into gluconic acid, which decreases pH and consequently protonation of the polymer chain. Thanks to coulombic forces, swelling occurs, releasing entrapped dye. This behavior may be used in the detection and drug delivery. The mentioned treatment may be considered for diabetes for the detection of glucose in the blood. 2O2 . . 123]. LThiobacillus thioparus, used as the recognition element, which was immobilized on sodium alginate and an agarose bed. Oxygen reduction was considered the detection sign. The authors confirmed that the obtained final biosensor had a satisfactory value of oxygen taken up by the immobilized cells.Trichloroethylene (TCE) is one of the common pollutants of groundwater and soil that is uneasy to degrade by microorganisms. The literature  reports 2+, Cd2+, Ni2+, Pb2+) ..33].The use of a sensor in dressings allows for control of the wound-healing process. The control may concern various important parameters of the wound, changes may indicate the healing stage, the presence of pathogens and necrotic processes. The sensor can continuously monitor the condition of the wound, which is especially important in the case of difficult wounds, such as ulcers, bedsores and diabetic foot. Monitoring reduces unnecessary and excessive changes in the dressing, which may affect, for example, fluctuations in moisture, irritation of tissues, and loss of barrier . Table 3The most frequently monitored physical parameter is pH. To monitor this parameter, scientists are developing indicators with different modes of operation, e.g., colorimetric and electrochemical indicators . Nischwitz et al.  producedAs pH stimulates volumetric changes, the amount of water present in the polymer matrix differs from 20% to 99% based on the pH that the hydrogel is immersed in. The main key to exhibiting pH sensitivity is the presence of a backbone polymer that contains weak acidic or basic groups, which become more ionized based on changes in pH. Depending on the pH of the solution the number of carboxylic ions changes, which influences the refractive index of the hydrogel . Different variations in sweat pH can be correlated with different pathological and physiological conditions. Usually, sensitive elements are trapped in a polymeric network (hydrogel). The responsiveness can be triggered by external influence for example reversible swelling or shrinking, which is caused by changes in the equilibrium of electrostatic forces. This is also sensitive to changes in pH, which also alternates caused by different diseases. Such a responsive material can be prepared by co-polymerization of 10 kDa poly(ethylene glycol)-diacrylate (PEG-DA) macromer with 2-carboxyethyl acrylate (CEA), resulting in a soft copolymer and, more importantly, it is pH sensitive. pH sensitivity is due to the carboxylic group of CEA, which is protonated in acidic conditions and deprotonated in a basic environment. Resulting in repulsion or attraction of chains. Healthy skin exhibits slightly acidic properties and the pH of the wound varies from alkaline to neutral around 7.15 to 8.93. Tamayol conducted experiments on alginate-based microfibers that are loaded with pH-responsive silica beads that change color, depending on the pH dye. The presence of hydrogel in healing wounds is beneficial as it maintains a moist environment. Changes in dressing color allow for monitoring of the healing process. Next to monitoring, it was determined that it is possible to enhance wound healing. Hydrogel made of poly(N-isopropyl acrylamide-co-acrylic acid) via radical copolymerization which was loaded with bovine serum albumin (BSA), which fills the role of growth factor, was constantly released at increased pH. Such a hydrogel may as well be loaded with antibiotic agents, and combined with the monitoring of bacterial infections with dye, results in a very effective way to enhance wound treatment. Kwon et al. fabricated hydroxyethyl cellulose hydrogel with hyaluronic acid for treating skin lesions. Based on swelling and shrinking properties thanks to changes in pH, the primarily captured drug was slowly released. The efficiency of such drug release was estimated to be more than 70% at pH 7.pH-sensitive hydrogels have also been investigated in peptide drug delivery, which cannot be delivered in acidic regions. Consequently, hydrogel protection is a chance of successful delivery, without losing the drug. For this purpose, Yadav and Shivakumar used soluble water chitosan to deliver the drug to the intestines. It was found that the sensitivity of the hydrogel was strongly connected to the concentration of the crosslinking agent, which was carboxymethyl chitosan and Carbopol. This method was used in the prolonged releasing theophylline in the intestine for treating nocturnal asthma. A similar treatment was developed by Dai et al. for delivering the anticancer drug as the pH close to the tumor decreases below the physiological range. It was also proved that NIR irradiation can accelerate drug release. Considered for this role were two polymers: N-(2-hydroxypropyl) methacrylamide (HPMA) and DF-PEG-PAHy/BPNSs. Polyethylene glycol dimethacrylate (PEGDMA) and methacrylic acid (MAA) in a molar feed ratio of 1:2 with ammonium sulphate result in pH-sensitive hydrogel and near-infrared (NIR) light and showed ideal mechanical and swelling properties. The material was tested in vivo as drug delivery for chemotherapy treatment (doxorubicin (DOX)) the drug was efficiently contained in the hydrogel structure for cancer therapy and then released by changes in pH or NIR. Composite consisting of graphene oxide and para-aminosalicylic acid after lyophilization results in the air-dried hydrogel. This combination of polymers exhibited both pH-sensitive properties and different drug release profiles in neutral and acidic conditions. It is sensitive to changes in the blood pH as well as the internal pH of infected macrophages . The future of biopolymer composites with indicators that respond to a change in the biological environment seems to be still a long way off. Scientific disciplines permeate and complement what creates a space of great potential . The netOne of the driving challenges in developing new indicator composites is an aging society. All countries face major challenges to ensure that their health and social systems will be ready for demographic shifts. Every country in the world is experiencing growth in both the size and the proportion of older persons in the population. WHO warns of the global demographic trend, the pace of population aging is much faster than in the past. Between 2015 and 2050, the proportion of the world’s population over 60 years will nearly double from 12% to 22%. Statistical data clearly shows that in the EU we currently have a trend of an aging society .The aging society will require the introduction of new medical technologies in hospitals and domestic use. Biosensors will transmit data from the patient’s home to the medical center, where they will be verified. Home care will be supervised, and the sensor system will send this information to the medical center in the event of any changes. The patient will be under constant supervision in a more favorable home environment. The future of biosensors will allow for extensive control of parameters at home and quick reaction of the control center to unfavorable changes The control of the environment by biosensors will be highly desirable in the future. In 2022, an ecological disaster took place on the Odra River. Currently, golden algae are indicated as the indirect cause of the catastrophe. The immediate cause was the increase in salinity. However, the problem is more complex and is the result of several stress factors, all of which were caused by human activity. The disaster caused mass mortality in fish, mussels and water snails in August 2022. Now the biomass of the perished animals is being decomposed by bacteria, which is still dangerous for the river . BiosensBiopolymers are of great importance in the design of biocomposites with sensors for various applications, for example, to be used in medicine or the environment. The variety of properties of biopolymer materials allows for wide application possibilities in many branches of industry for example biosensors used in medicine. Biosensors are fast, portable systems that enable monitoring of the environment in which they are located. These materials play an important role in medical diagnostics and patient monitoring. Biosensors are suited to various diagnostic and real-time detection challenges owing to the use of biological molecules, tissues, and organisms. Medical biosensors are suitable for monitoring health parameters. These medical materials help biomedical engineers, researchers, molecular biologists, oncologists and clinicians with the development of point-of-care devices for disease diagnostics and prognostics. It also provides information on developing user-friendly, sensitive, stable, accurate, low cost and minimally invasive modalities which can be adopted from the lab to clinics.Civilization diseases, i.e., diabetes, strokes, and diseases of the cardiovascular system, along with an aging society, require the use of new medical therapies. A future direction is composites based on biopolymers with sensors informing about physiological and chemical aspects. Such solutions will enable faster and more effective treatment of difficult-to-heal wounds, such as pressure ulcers, diabetic foot ulcers and wounds after extensive surgeries. They are necessary to improve patients’ functioning and survival. Scientists have conducted numerous studies and the development of medicine has long exceeded the wildest expectations of science fiction authors. This level of science allows us to design very advanced sensory technologies. The only barrier is the economic aspect, such materials are expensive and difficult to access. Nevertheless, their development is certain, and the costs will be reduced in the future. It is difficult to calculate human health and life, and therefore medical care should use new technologies.