Traditional Uses of Animals in the Himalayan Region of Azad Jammu and Kashmir

Background: The use of animals and animal-derived products in ethnopharmacological applications is an ancient human practice that continues in many regions today. The local people of the Himalayan region harbor rich traditional knowledge used to treat a variety of human ailments. The present study was intended with the aim of examining animal-based traditional medicine utilized by the population of the Himalayan region of Azad Jammu and Kashmir. Methods: Data were collected from 2017 to 2019 through individual and group interviews. Data on traditional uses of animal products were analyzed, utilizing following indices such as the frequency of citation, use value, relative importance, similarity index, principal component analysis, and cluster analysis to find the highly preferred species in the area. Results: Ethnomedicinal uses of 62 species of vertebrates and invertebrates were documented. Flesh, fat, bone, whole body, milk, skin, egg, head, feathers, bile, blood, and honey were all used in these applications. The uses of 25 animals are reported here for the first time from the study area (mainly insects and birds, including iconic species like the kalij pheasant, Lophura leucomelanos; Himalayan monal, L. impejanus; and western tragopon, Tragopan melanocephalus). The diversity and range of animal-based medicines utilized in these communities are indications of their strong connections with local ecosystems. Conclusion: Our results provide baseline data valuable for the conservation of vertebrate and invertebrate diversity in the region of Himalayan of Azad Jammu and Kashmir. It is possible that screening this fauna for medicinally active chemicals could contribute to the development of new animal-based drugs.


INTRODUCTION
Zootherapy is described as the use of animals or animal-derived materials to treat human ailments (Costa-Neto, 2005;Holennavar, 2015;Ahmad et al., 2021). The use of animals with medicinal properties continues to be a common practice worldwide. Zootherapy techniques and materials are utilized in traditional and nanomedicine for the treatment of different diseases (Kassam, 2002;Lawal and Banjo, 2007;Prakash and Verma, 2021). It is documented that almost 13% of the drugs used by traditional Chinese medicine are derived from vertebrates and invertebrates (Still, 2003). In Ayurvedic medicine, 15-20% of drugs contain vertebrate and invertebrate products (Unnikrishnan, 1998). In Tibetan medicine, the products of vertebrates and invertebrates are utilized in more than 111 drugs (Singh, 2000).
Many societies are rapidly losing their ethnopharmacological knowledge; so, documenting this knowledge before it is lost is increasingly important Löki et al., 2021;Mandal and Rahaman, 2022). Likely because of the dominance of plants in traditional medicine systems, the use of animals and animal-derived products in traditional medicine has been underdocumented. Pakistan has a rich faunal diversity, including 195 species of mammals (Roberts, 1997), 668 species of birds (Mirza and Wasiq, 2007), 195 species of reptiles (Khan, 2006), and 24 species of amphibian studied by Khan (2010). To date, a number of studies have documented the use of animal parts in traditional medicine in different parts of Pakistan Adil and Tariq, 2020;Aslam and Faiz, 2020;Mughal et al., 2020;Noor and Haider, 2020;Haidar and Bashir, 2021;Ijaz and Faiz, 2021;Ijaz and Iftikhar, 2021;Saleem et al., 2021); however, ethnomedicinal uses of animals in Azad Jammu and Kashmir have never been reported.
Animals and its derived products are important elements in many traditional treatments (Ferreira et al., 2010;Albuquerque et al., 2012;Altaf and Faiz, 2021), and they have presumably utilized since prehistoric times . Traditional information can lead scientists to promising natural sources of new medicines, making it a powerful ally in the discovery of new drugs (Saleem et al., 2021;Habib, 2022). A suitable model for replicating contact dermatitis is phenol-induced ear edema. When phenol comes into direct contact with the skin, "keratinocytes" release chemical mediators that are crucial in prime contact irritation reactions, including as pro-inflammatory cytokines (Lim et al., 2004). These pro-inflammatory cytokines are made in a different way than those synthesized by PKC (as occurring in inflammations induced by croton oil). The rupturing of the "keratinocyte plasma membranes", which leads to the liberation of pre-formed IL-1, as well as other inflammation mediators, is thought to be the cause of cutaneous irritations (Murray et al., 2007).
Zoonotic diseases are transferable diseases caused by infectious agents (such as viruses, bacteria, prions, or parasites) that can be transferred from a non-human animal to a human. Zoonotic diseases have caused a series of major global public health issues (malaria, yellow fever, avian flu, swine flu, West Nile virus, MERS, SARS, etc.), culminating in the current coronavirus health crisis (Altaf, 2016;Altaf, 2020). Different pathogens have different modes of transmission (Kruse et al., 2004;Van Vliet et al., 2017), so the risk of zoonotic diseases depends on the type of animals with which humans are in contact (as well as the duration and nature of contact) (Bilal et al., 2021). For example, the prevalence of diseases from fish to humans is very low (EHS, 2016b), while the risk of transmission from amphibians is higher due to human sensitivity to their porous skin (EHS, 2016a). The main aim of this study is to determine what animals local populations in Pakistan are in contact with in order to contribute to an understanding of the risk of zoonotic disease transmission due to ethnopharmacological uses of animals.
Human impacts on natural systems are complex. Many indigenous cultures have traditionally promoted ways of life that are relatively balanced in relation to the sustainability of their resource use. On the other hand, the forces of capitalism coupled with a conceptual nature-culture divide and propagated through the global spread of colonialism have resulted in extractive approaches to resource use that threaten the resilience of the majority of ecosystems. Ethnozoological research is critical to understanding the sustainability of biocultural systems (Fopa et al., 2020). Cultural uses of animal species (i.e., food, hunting, medicine, entertainment, religious practice, and trade) may promote beliefs and behaviors that help to conserve these animal species; however, if they are practiced unsustainably, or affected by commercialization or other political and economic factors, they may negatively affect or even endanger these animals. The use of animal species for traditional medicine and cultural purposes by local communities must also be considered in relation to other factors, such as changes in climate and habitat (Alves, 2012;Alves et al., 2018). There exists a global need to find new approaches to dealing with the present crisis of biodiversity loss (Boivin et al., 2016), and ethnozoology provides critical insights into the practices of local communities, allowing conservation efforts to effectively partner with resource stewards to promote the overall integrity of biocultural systems (Saunders, 2003;Dickman, 2010). This study on the medicinal uses of fauna by the rural and urban people of the Himalayan region of Azad Jammu and Kashmir is part of a broader research project to document the uses of animals by local communities throughout Pakistan (Muhammad et al., 2017a;Muhammad et al., 2017b;Muhammad et al., 2017c;Altaf et al., 2018;Altaf, 2020).

Description of the Study Area
Individual and group interviews were conducted in six different sites of the Himalayan region in Azad Jammu and Kashmir during 2017-2019 ( Figure 1). The study area is located between 33°and 35°North latitude and 73°and 75°East longitude, in the foothills of Himalayas on the North East side of Pakistan, with an average elevation of 6,325 m in the north and 360 m in the south . Azad Jammu and Kashmir (AJ&K) is a cultural and geographical land of narrow, long, strip and occupies an area of 13,297 km 2 , with >4 million population. The main rivers of AJ&K are Jhelum, Poonch, and Neelum. The climate of this region is subtropical with an average rainfall of >150 cm. Spruce (Abies pindrow), Kail (Pinus excelsa), cheer (Pinus willichiana), deodar (Cedrus deodara), fur (Pinus spp.), and some other conifer species are dominant trees in AJ&K forests (PM, 2008;Ch et al., 2013;Khan et al., 2017;FWFD, 2021).
The study area is dominated by different tribal groups, such as Khawaja, Gujjar, and Rathor, which are the most common. Pahari, Kashmiri, and Gujjari are the major languages spoken, while Urdu is the official language, which is spoken as a second language by most people. The population of Haveli District was 112,000 in the census of 1998 and 157,000 in the census of 2015. The density was 262 people per sq. km in 2015. The average household size in the district is around 7, with most people living in joint family structures. The majority of the population lives in rural areas and is entirely Muslim. Most of the people (˃70%) in the study area are educated .

Data Collection and Analysis
Before beginning fieldwork, consent was obtained from the "Department of Zoology, Women's University of Azad Jammu and Kashmir, Bagh-Pakistan," while questionnaires and interviews were arranged to record the ethnomedicinal uses of animals. Data were taken from respondents (n = 210) who included government employees, housewives, students, farmers, drivers, and customary wellbeing practitioners (Supplementary Table S1). Respondents were chosen based on their having basic awareness of folk medicines of wild animals. During the field survey, prior informed consent was obtained from each participant, and general standards/guidelines of the International Society of Ethnobiology (ISE) (http://www.ethnobiology.net/) and Consensus Statement on Ethnopharmacological Field Studies (ConSEFS) (https://www.journals.elsevier.com/journal-ofethnopharmacology/) by Heinrich et al. (2018) were followed.
Field guides of mammals, birds, and herpetofauna "Mammals of Pakistan" (Roberts, 2005), "Birds of Pakistan" (Roberts, 1991(Roberts, , 1992, and "Amphibian and Reptiles of Pakistan" (Khan, 2006) were shown to informants to verify which species they described. Basic data on medicinal uses were then used to generate different indices including "frequency of citation," "use value," and "relative importance," which were then analyzed using statistical methods including "similarity index," "principal component analysis," and "cluster analysis."

Quantitative Analysis
The ethnozoological data were analyzed by various indices, which include "FC" (frequency of citation), RI (relative importance), and UV (used-value).

Frequency of Citation and Relative Importance
The frequency of citation is the number of respondents who described the medicinal uses of wild fauna species. The relative importance index was intended by the formula, as reported by Oliveira et al. (2010).

RI PP + AC
where PP stands for pharmacological property quantity and AC is the maximum number of ailment categories treated by the most resourceful species divided by the number of ailment categories treated by a given species.

Use Value and Similarity Index
The use value (UV) is the quantitative measure of the relative importance of specific animal species known locally. UV and the SI were calculated following the method reported previously (Trotter and Logan, 1986;Phillips and Gentry, 1993), using the formula:

UV U n
The number of citations per species is n, and the number of informants is U.
Note: S a = Similar documented ailment in the previous and present studies, T a = Total documented ailment in the present study.

Statistical Analysis
Data were analyzed in "Microsoft Excel 2010" (Microsoft, Redmond, WA, United States), whereas inferential statistical analysis was performed by using R software 3.6.3 and PAST 3.20 (Hammer et al., 2001). In addition, traditional uses of the body part(s) of animal species and their mode of application were represented in chord diagrams generated with the "circlize package (24)" in R software 3.6.1 (Gu et al., 2014).
Thirty-nine diseases were treated with different animal parts and products (Figure 3), such as flesh, bone, whole body, milk, skin, egg, head, feather, bile, blood, and honey. Flesh was the most consumed (n = 35) body part, followed by bone, whole body, milk, skin, egg, head, feather, bile, blood, and honey ( Figure 4). Local inhabitants use the fat of different species such as little egret (Egretta garzetta) and cattle egret (Bubulcus ibis) to treat memory and epilepsy, golden eagle (Aquila chrysaetos) to treat wound healing, and regulate blood chemical, Alexandrine parakeet (Psittacula eupatria) to treat memory, great tit (Parus major) to treat male impotency and skin problem, duck (Anas platyrhynchos domesticus) to treat kidney problems, heart problems, BP, male impotency, piles, blindness, and eyesight, Asiatic black bear (Ursus thibetanus) to treat joint pain and male impotency, Indian crested porcupine (Hystrix indica) to treat joint pain, Asiatic jackal (Canis aureus) to treat skin problems, Hazara gauk (Duttaphrynus melanostictus) to treat antibacterial and antifungal, agror agama (Laudakia agrorensis) to treat joint pain, backbone pain, and male impotency, brown cobra (Naja oxiana) to treat joint pain, piles, and eyesight, oriental garden lizard (Calotes versicolor) to treat joint pain, and leopard gecko (Eublepharis macularius) to treat cancer ( Figures 5, 6). Likewise, the meat of snow partridge, Lerwa lerwa, was used to treat fever; western tragopan, Tragopan melanocephalus, was used to treat lung problems and weakness; common quail, Coturnix coturnix, was used to treat bilious, heart problems, TB, joint pain, backbone pain, and paralysis; rain quail, Coturnix japonica, was used to treat and regulate blood chemical; chukar partridge, Alectoris Chukar, was used to treat weakness; black francolin, Francolinus francolinus, was used to treat joint pain and lung problems; grey francolin, Francolinus pondicerianus, and Himalayan monal, Lophophorus impejanus, were used to treat weakness; kalij pheasant, Lophura leucomelanos, was used to treat weakness, fever, and memory; common pigeon, Columba livia, was used to treat Parkinson's disease, ptosis, and tongue problem; hill pigeon, Columba rupestris, was used to treat wound healing; spotted dove, Spilopelia chinensis, and oriental turtle dove, Streptopelia orientalis, were used to treat paralysis and enhance muscle power; Asian koel, Eudynamys scolopaceus, was used to treat spleen problem; oriental scopus owl, Otus sunia, was used to treat whooping cough; common hoopoe, Upupa epops, was used to treat stomach problems, liver disease, bladder disease, and eyesight; Asian house martin, Delichon dasypus, was used to treat male impotency; barn swallow, Hirundo rustica, was used to treat male impotency; streaked laughing thrush, Trochalopteron lineatum, was used to treat weakness; common myna, Acridotheres tristis, was used to treat whooping cough; russet sparrow, Passer cinnamomeus, was used to treat paralysis, male impotency, and liver; grey wagtail, Motacilla cinerea, white wagtail, Motacilla alba, and citrine wagtail, Motacilla citreola, were used to treat kidney problems; duck, Anas platyrhynchos domesticus, was used to treat kidney problems, heart problems, BP, male impotency, piles, blindness, and eyesight; hen, Gallus gallus domesticus, was used to treat kidney problems, heart problems, weakness, memory, eyesight, male impotency, diabetes, stomach problems, and BP; alpine musk deer, Moschus chrysogaster, was used to treat paralysis; Rhesus Macaque, Macaca mulatta, was used to treat wounds; red fox, Vulpes vulpes, was used to treat male impotency; giant red Himalayan squirrel, Petaurista petaurista, was used to treat diabetes; cow, Bos taurus, was used to treat and enhance protein, weakness, and boil; and Bubalus bubalis was used to treat fever and enhance protein (Figures 5, 6).

Principal Component Analysis and Cluster Analysis
Statistical analysis with the assistance of "PCA" showed that the first two axes of the "PCA" has 100% variation and "PC 1" and "PC 2" have 98.5 and 1.5% variations, respectively (Figures 8, 9). Variables loaded onto the x-axis "PC 1" includes FC (r = 0.99454),  (Figures 8, 9). The results of PC1 exhibit positive correlations between FC, UV, and RI, while that of PC2 indicate a negative correlation with FC and a positive correlation with UV, as well as RI variables. The analysis demonstrated that two groups are noted in the "cluster analysis" in the Himalayan region, AJ&K, i.e., "group one" (G1) and "group two" (G2). "G1" and "G2" have a similarity of almost 0.8 points. G1 is further divided into two groups known as SG1-I (subgroup 1-I) and SG1-II (subgroup 1-II), while both have a similarity of approximately 0.5 points. Likewise, G2 is further divided into two groups known as SG2-I (subgroup 2-I) and SG2-II (subgroup 2-II), while both have a similarity of about 0.6 points. SG1-II has the following species of animals with diseases coded as COEP, COPH, HNBP, HNMI, HNKP, HNEM, and HNSM; while SG1-II has the following BFFV, BFEP, DUHP, DUKP, HPWH, and CPPD ( Figure 10).

DISCUSSION
In ethnozoological research, socio-demographic data on respondents (age, gender, occupation, ethnicity, and education) are incredibly useful, as this component plays a key role in interpreting and analyzing the feedback received (Easthope, 1995;Hanif et al., 2019). Male respondents made up 94% contribution, whereas female respondents were rare in the present study. This is because most of the females are housewives and do not meet with strangers, so more males are selected for interviews. Altaf et al. (2017) discovered similar results in a research of ethnomedicinal and cultural activities of mammalian and avian in the region of Punjab, Pakistan. In fact, males hunted animals for food as well as for medicine, which could explain our findings. Additionally, the informants in village areas had more knowledge and information regarding the use of species for human ailments when compared to the informants in urban regions. These results were alike to earlier information from the district of Negev, Israel (Friedman et al., 1986).
The inhabitants of the region of Himalayan in AJ&K reported the ethnomedicinal uses of 62 animal species to treat 39 different diseases including male impotency, weakness, joint pain, memory loss, paralysis, piles, eyesight, stomachache, whooping cough, liver, and kidney problems among others in the present study. Similarly, 32 animal species, invertebrates and vertebrates, for treating 37 types of ailments were reported in southern regions of KPK, Pakistan. The major treated ailments were night blindness, epilepsy, cancer, hepatitis, asthma, paralysis, whooping cough, and brain hemorrhage (Mussarat et al., 2021). They reported the use of Gallus gallus domesticus for joint pain, blood pressure, weakness, hepatitis, diabetes, Capra hircus for hepatitis C, night blindness and joint pain, Passer domesticus for abdominal pain, and Ovis aries for the regulation of blood level, which supports our findings.
COEP (flesh of Bos taurus enhances the amount of protein) and COPH (milk of Bos taurus is used to treat weakness) were documented as the most often consumed with FC = 29 in the Himalayan region of Azad Jammu and Kashmir. Most animals were versatile in the context of utilization (RI = 3.45), such as CQB (flesh of Coturnix coturnix is used to treat bilious), CQJP (flesh of Coturnix coturnix for joint pain), CQBP (i Coturnix coturnix is used to treat backbone pain), CQPL (flesh of Coturnix Coturnix is used against paralysis), and CPPD and HPWH (flesh of Columba livia and C. rupestris is used to treat Parkinson's disease). The maximum relative importance is an indication of high accessibility and affordability of a species (Umair et al., 2019). Animal species with high RI values could be focused to evaluate their pharmacological and therapeutic potential. Therefore, statistical analysis is of significant value in ethnobiological studies because it facilitates the researcher in the selection of appropriate species and their body parts for chemical profiling and pharmacological/clinical studies. The ethnopharmacological data were calculated through "PCA," which allocated for the ordination of designs in terms of three variables, i.e., FC, UV, and RI. Statistical analysis with the help of "PCA" showed that the first two axes have 100% difference and "PC 1" has 98.5% and "PC 2" has 1.5% variations. These findings were in agreement with other studies (Altaf, 2020).

Novelty of Data
The folklore is an animal-based-medicinal concept of populations of the region of Himalayas, AJ&K. It means that people have a strong association with the ecosystem. For the first time, medicinal uses of animals from Azad Jammu and Kashmir were investigated. Furthermore, applications of 49 animal species are used to cure different diseases in humans. Single species of earthworm also has a zero similarity index. Flesh, fat, bone, whole body, milk, skin, egg, head, feather, bile, blood, and honey were utilized as body parts. Lerwa lerwa, Tragopan melanocephalus, Coturnix japonica, Alectoris chukar, Francolinus pondicerianus, Pucrasia macrolopha, Lophophorus impejanus, Lophura leucomelanos, Bubulcus ibis, Neophron percnopterus, Psittacula eupatria, Otussunia, Parus major, Delichon dasypus, Hirundo rustica, Motacilla cinerea, Motacilla alba, Motacilla citreola, Petaurista petaurista, Paraconophyma spp., Meranoplus bicolor, Actiasselene, Luciola substriata, Androctonus spp., and Libythea lepita were noted for the first time from Himalayan region, AJ&K. This study gives information that could be useful in the conservation of animal biodiversity in Azad Jammu and Kashmir's Himalayan region. For wild animalbased new pharmaceuticals, the screening of medicinal-active compounds and either "in vivo" or "in vitro" examination of biological activities of fauna with maximal "FC," "UV," "RI," and "SI" could be relevant.

CONCLUSION
To the best of our knowledge, ethnomedicinal uses of the diverse fauna of the Himalayan regions of Azad Jammu and Kashmir have rarely been reported before. Our findings revealed that local inhabitants have strong associations with animal species in their surrounding environment and use them in their primary healthcare system to treat various diseases. In addition, medicinal uses of more than 60% of the species were reported for the first time from this area. Animal species with high medicinal values should be further explored for bioactive compounds and in vitro/in vivo activates to introduce novel animal-based health-care products. Bos taurus was documented as the most often consumed with FC = 29, while Coturnix Coturnix and Columba livia were documented to be highly versatile in their utilization (RI = 3.45) in the Himalayan region of Azad Jammu and Kashmir.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The present study focused on traditional uses of animal species by the local inhabitants of the Himalayan region of Azad Jammu and Kashmir. During the field survey, prior informed consent was obtained from each participant for the documentation and sharing of information. General standards/guidelines, ethical norms, and rules of the International Society of Ethnobiology (ISE) (http://www.ethnobiology.net/) and Consensus Statement on Ethnopharmacological Field Studies (ConSEFS) (https://www. journals.elsevier.com/journal-of-ethnopharmacology/) were followed.

AUTHOR CONTRIBUTIONS
MF performed field survey and formal analysis and wrote the original draft. MA and AA supervised the project, provided intellectual support and resources, was involved in the methodology and data analysis, and edited and revised the final article. MU helped in data analysis and reviewed the article. KA and YE provided financial assistance and was involved in the revision and editing.

FUNDING
This research project was funded by the "Researcher Support supporting Project" at "King Saud University" via Researcher Support Project no. RSP/2021/189.