Ethno-medicinal uses of vertebrates in the Chitwan-Annapurna Landscape, central Nepal

Traditional knowledge on the use of animal products to maintain human health is important since time immemorial. Although a few studies reported food and medicinal values of different animals, a comprehensive ethno-medicinal study of vertebrates in Nepal is still lacking. Thus, present study is aimed at documenting the ethno-medicinal knowledge related to vertebrate fauna among different ethnic communities in the Chitwan-Annapurna Landscape, central Nepal. Data was collected by using semi-structured questionnaires and analyzed by using Use Value (UV), Informant Consensus Factor (ICF) and Fidelity level (FL). Results showed a total of 58 (53 wild and 5 domestic) species of vertebrate animals. They were used to treat 62 types human ailments. Four animals were also used for veterinary diseases and agriculture benefits. The most widely used species was Felis chaus (UV = 0.25) with 3 use-reports by 10 informants. Cardiovascular and dental problems had the highest ICF value (0.974) with cardiovascular problems having 351 use-reports for 10 animal species and dental problems having 77 use-reports for 3 animal species. The least ICF was found in ophthalmological problems (ICF = 0.833, use reports = 7 for 2 species). We concluded that the different animals were an important part of traditional medicine for the local people living in the Chitwan-Annapurna Landscape. However, the majority of animals and most likely to be threatened due to their uses. The present documented ethnozoological knowledge can be used in conservation and management of vertebrates so that they could be protected for future generations.

in Traditional Chinese Medicine in China [37]. There is also a trade of high number of traditionally used natural products by various ethnic and cultural groups for the treatment of various ailments [3].
To the greater extent, many traditional medicines are traded by many big pharmaceutical companies around the world. World Health Organization (WHO) has selected a total of 252 essential chemicals to prepare drugs, where animals alone contribute 8.7% [6].
A large number of animals are used as medicine under traditional systems such as Ayurveda, Unani, Homeopathy, and Tibetan traditional medicines [4][5][6]. Many studies have shown use of animals as medicine in Nepal [5,[7][8][9][10][11][12][13] including a few studies from Chitwan-Annapurna Landscape, but are mainly related to medicinal plants [14][15][16][17] and wild edible plants [18]. Chitwan-Annapurna Landscape lies in between Chitwan National Park in the south and Annapurna Conservation area in the north with high variation in elevation (150 m to 8000 m). This area is mostly inhabited by many types of indigenous and multi-cultural ethnic groups. Therefore, such an area is important for studies related to traditional medicine.
Nepal comprises 125 different multicultural ethnic groups with more than 123 different languages [19]. Thus, Nepal is well-known for rich cultural heritage with diverse ethnic groups [9]. In most ethnic groups, indigenous knowledge on uses of different animals in medicine is generally passed verbally from one generation to the next generation. Such knowledge is commonly lost with the demise of knowledgeable person [2,11,20]. It is, thus, important to systematically document such indigenous knowledge so that it can be protected for future generations. This study is aimed to collect and

Study area
The study area located in the Chitwan-Annapurna landscape, central Nepal (Fig. 1). The region is connected with the Chitwan National Park in the south and Annapurna Conservation Area in the north.
This landscape is rich in globally outstanding biodiversity including three Word Wildlife Fund (WWF) Global 200 Eco-regions (Terai-duar Savanna and Grasslands, Himalayan Subtropical Broadleaf Forests, Alpine Shrubs and Meadows), and two Ramsar sites (Beeshazari lake, Chitwan and Lake Clusters of Pokhara valley, Kaski) [21].

Data collection
For our data collection, we divided the whole study area into four different study blocks based on topography ( Fig. 1 The ethno-medicinal data on uses of animals (mainly vertebrates) was collected by using Participatory Rural Appraisal (PRA) method [20,[22][23][24]. In this method we used sets of questions related to use of animals as ethnomedicine and discussed their local status and medicinal properties. For that purpose, we used the photographs of the vertebrates present to that area and discussed in group. The local healers, medicinal practitioners, teachers, social workers were involved in the PRA. We also considered a fair gender composition of the group.
Consent was taken from the respondent prior to formal interview. Semi-structured questionnaires were also used to obtain information from the local people [25,26]. The respondents were chosen randomly, but were well represented from different ethnicity, geographic locations, age, sex, profession and education levels [27]. Total of 204 people were interviewed during 2017-2019. We used photographs and images of different vertebrates when conducting interviews. The detailed information including local name of the animals, parts used, methods of preparation and mode of administration were recorded. We also documented vernacular names, methods of preparation and the doses of medicine used against different ailments. Latin names and classification of the animals were obtained from standard literature [28][29][30][31][32]. We also recorded conservation status of each animal by using above mentioned literature and IUCN Red data book [60].
Based on the information obtained from informants in the study area, all the reported human related ailments were grouped into 11 categories (Table 1) viz. cardiovascular problem, dental problem, dermatological problem, ear, nose and throat problem, gastro-intestinal problem, musculoskeletal problem, neurological problem, ophthalmological problem, reproductive problems, respiratory problem and others (fever and headache). In addition to this, we also recorded the veterinary and agriculture uses.

Informant consensus factor (ICF)
To see if there was agreement in the use of animals in the ailments categories between the animal users in the study area, we used the informant consensus factor (ICF) [33,34].
The informant consensus factor (ICF) for ailment category c was calculated as ICF c = (Nur j -Nt j )/(Nur j -1), where Nur j is the number of use-reports in each ailment category c and Nt j is the total number of taxa used in each ailment category c by all informants.
In each case if an animal was mentioned by an informant as 'used' then we considered it as one 'usereport.' If one informant used an animal to treat more than one ailment in the same category, we The ICF ranges from 0 to 1, where high values (close to 1) are obtained when only one or a few animal species are reported to be used by a high proportion of informants to treat a particular ailment meaning that there is a narrow well-defined group of animal species used to cure a particular ailment category and/or that information is exchanged between informants. On the other hand, low ICF values (close to zero) indicate that informants disagree over which animal to use due to random choosing or lack of exchange of information about use among informants [62].

Fidelity level (FL)
To determine the most frequently used animal species for treating a particular ailment category by the local people of the study area, we calculated the fidelity level (FL) [63]. For each species s and each ailment category c, we calculated the value FL sc using the following formula: where Np sc is the number of use-reports cited for a given animal species s for a particular ailment category c and N s is the total number of use-reports cited for any given species s. The animal species with the highest FL sc value is considered the most preferred species for ailment category c.

Use value (UV)
The relative importance of an animal species used as medicine in the study areas was calculated with the help of the use value (UV) for species s [64]: UV s = ∑U s /N s where U s is the number of use-reports cited by each informant for a given animal species s and N is the total number of informants interviewed for a given animal species s. Use values are high when there are many use-reports for an animal and low when there are few reports related to its use.
To determine correlations between FL value and UV values for each animal species, we used Spearman's correlation coefficient because data were not normally distributed.

Demographic details of informants
A total of 204 informants (70 female and 134 male individuals, aged between 18 and 82 years) participated in the study. They belong to different castes (12) and communities (eight of Indo-Aryan or Tibeto-Burman language speaking groups). A large number of respondents were in between 50 to 59 years (n=53). This clearly showed that ethno-medicinal knowledge was higher in aged groups than in young groups. The main reason of little knowledge among young generation might be due to growing number of hospital facilities, migration of people to urban areas and abroad for study and employment, and also the influences of mixed cultures due to cross-cultural communications or settlements in new areas [35,36].
Mostly the people living in the villages have strong belief on the traditional healing system and traditional medicine. The male and female ratio may indicate the dominancy of the male ethnomedicinal practitioners than female. Such type of trends was also reported in different ethnomedicinal studies [2,10,14,36] (Table 2). About 43% of the respondents were farmers and healers who had broad knowledge of ethno-medicine. There were 38% of the respondents with basic level and 26% of them with secondary level education. Locally popular traditional healers and wizard doctors were involved in the focus group discussion.

Faunal diversity and uses
The present research revealed the use of 58 animal species of 23 orders, 37 families and 53 genera to cure 62 human and 3 veterinary ailments (Table 3). Among the medicinal animals, 53 animal species are collected from the wild and five are domesticated (Table 3). Among the used animals, two animals have poisonous property ( Table 3). The use of threatened wild animals was also reported in the treatment of different ailments (Table 3). It shows that many wild animals if used regularly would likely decline leading to possible extinctions.
The order, family, scientific names, English names, Nepali names, IUCN category, use value (UV), parts used, uses are presented in Table 3. Among 58 vertebrates, 24 species (41%) were Mammalia, 16 Aves (28%), 6 Reptilia (1%), 3 Amphibia (0.5%) and 9 Actinopterygii (1.5%) (Fig. 2). Local people and poachers from outside killed animals for their body parts have been causing the greatest threats to wildlife [20,38]. Similar types of threats were also reported during this study. Mammals considered as the most important vertebrate group that were used very high in traditional medicine. Rural people believed that wildlife mainly mammals are the sources of protein and other essential supplementary foods and medicines [39]. Similar studies showed the uses of vertebrates for more than 232 traditional zoo therapeutic remedies for human and animal health [12], and food and medicinal purposes of humans [10]. The use of a number of animals and the drugs derived from them recorded from the different altitudes of the study area. Practice of using traditional medicine was found higher in the mid hills and mountainous region than Terai region (Chitwan) ( Table 1). The local people used cooked meat of Golden jackal for treatment of paralysis and preparation of wine for treatment of rheumatism. Similar type of the practices also reported by Lohani [10] in Tamang community of Sindhupalchowk and Poudel and Singh [40] reported use of meat and fat of Golden jackal for treatment of the rheumatism in Darai community of Chitwan, Nepal. Similarly, Rai and Singh [13] reported such practices in Rai community of Bhojpur. Bones and meat of the animals are full of calcium, protein and phosphorous hence, soup of the bone and meat of animals is given for the person suffering from the muscular spasm, cramp, bone fracture, arthritis and energy (Table 3). A similar type of treatment methods was also recorded in the study of Lohani [10] in Tamang community, Nepal; Vijayakumar et al. [2] in Kerela adjoinig areas of Mt. Abu Wildlife sanctuary, India; Nijman and Shepherd [20] in Kyaiktiyo, Myanmar.

Veterinary important traditional medicines and insecticides
Medicinal fauna from four different animal families occurring in four genera and four species have veterinary importance. Generally, animal's parts and products such as urine, droppings, fat and meat are used. They are used internally or externally (Table 3). However, most of the people in the study area have little idea and knowledge about veterinary and agricultural use of vertebrates.
Furthermore, this study also indicated that more than 81% of vertebrate species used treating more than one ailment. Similar studies outside Nepal also reported the wide use of animals in ethnomedicines such as González et al. [41] recorded use of 30 wild vertebrates to treat domestic animals in Spain; Souto et al. [42,49] reported 11 animals for ethnoveterinary medicine in the semi-arid region of Northeastern Brazil and Gupta et al. [43] reported a total of 11 species of vertebrates for treating various veterinary diseases in India.

Animal parts used
The animal parts used for treating different ailments were of 22 types. Meat was the most preferred parts (n=48), possibly because meat has more protein and medical properties followed by fat (n=11), fecal matter (n=6), gallbladder, horn and antler and blood (each n=5), egg and claws (each n=3), urine, skin, milk, ghee and hair (each n=2), hoofs, feathers, teeth, brain, stomach, shell, quail, bone and legs (n=1) (Fig. 3, 6,7). Similar study of Quave et al. [12] reported whole animal, milk and milk products, meat/animal flesh, fat, honey and eggs, feces, urine, and seminal fluid in different treatment in different ethnic groups of Albania, Italy, Spain and Nepal. In previous studies, it was found that the ethno medicines are prepared from the animal parts and products [2,4,11,13,46,48].

Medical preparations and their admission
The medical remedies were based on many kinds of preparations ranging from a preparation made out of a single animal for a single ailment to use of animals in combination (Table 3). There were 12 types of preparations used in the study area. Cooked meat was commonly practiced (31%) followed by boiled and oil (each 12%), paste (11%), soup (10%), raw meat (9%), dry (6%), ash (3%), powder and lotion (each 2%) and fume and wind (each 1%) (Fig. 4). Consumption practices of raw body parts of animals are common for curing diseases in many ethnic groups at global level [2,23,35,40,44,45]. However, consumption of raw meat may increase the risks of transmitting different types of parasites and diseases to human [11,46].
The most common mode of admission of medicine is oral (67%) followed by topical application (30 %) and drop (4%). Topical use is an important way of remedy of musculo-skeletal problems like muscular pain, fractures, rheumatisms and arthritis. Such modes of administration were found in studies from Korea [46], in India [2] and also in Nepal [11,40].

Informant consensus factor, fidelity level and use value
The results of the informant consensus factor (ICF) calculation show that the value in our study ranges from 0.833 to 0.974. Cardiovascular and dental problems have the highest ICF value 0.974, with cardiovascular problems having 351 use-reports for 10 animal species and dental problems having 77 use-reports for 3 animal species. It is followed by musculoskeletal problems (ICF = 0.973; 926 use-reports, 46 species). The least agreement between the informants was observed for animals used to cure ophthalmological uses with ICF value 0.833 with 7 use-reports for 2 animal species (Table 4). Low ICF value might be due to lack of communication for the treatment of such ailments among the people of different cultures, different localities and ethnicities of the study area. Local people believed that there was no any side effects while using these ethnomedicines. Besides, this study helps to generate ethnozoological knowledge among the local people.
When selecting the most preferred animal species for each ailment category, we took the highest FL (%) in each category of ailment (  (20 %) for Ophthalmological problem purposes. The 100% animals indicate that healers and local respondents were used that animals for the treatment of same disease. It implied that well-known species were used more than the little-known species to cure the disease or disorders [24,46,47] The most commonly used species was Felis chaus (UV = 0.25) with 3 use-reports by 10 informants. It was followed by Panthera tigris (UV = 0.23) with 6 use-reports by 57 informants, Rhinoceros unicornis (UV= 0.16) with 6 use-reports by 43 informants and Columba livia (UV= 0.14) with 3 use-reports by 22 informants (Table 3).
The correlation between the highest fidelity level (%) in ailment categories and animal use value (UV) was not significant (Spearman's correlation test: r 2 = 0.038, p = 0.209) indicating that the animals systematically used for a specific ailment category are not necessarily those used commonly in the region. Although animals with high FL or UV are the most preferred species in study sites (Table 4 and 5), animals with low FL or UV should not be neglected as failing to mention them to the future generation could increase the risk of gradual disappearance of the knowledge.

Conservation status
Due to lack of modern medical facilities and belief on the traditional healing system, some people of the study area forced to use animals and their body parts for curing various ailments. Besides, superstitions and mythologies were very popular among the ethnic groups that may also play vital roles for using the animals. Therefore, they intentionally or unintentionally kill animals for ethnomedicinal uses which might increase threats to wildlife including many Globally Threatened Species.
Out of total (Table 3). Ten animals were Globally Threatened (2 Endangered, 8 Vulnerable), five were Near Threatened, one was Data Deficient and 37 Least Concerned species according to IUCN Red list [60] (Fig. 5). The Government of Nepal implemented National Park and Wildlife Conservation (NPWC) Act 1973 and Forest Act 1993 to protect wildlife and their habitats in Nepal. These laws strictly prohibited the hunting and killing of wildlife. There is no permission of killing the wildlife listed in CITES for food, medicine, and their trade. However, sometimes these laws malfunction due to local religious norms and cultural believes enable them to kill those animals. The tribal people have scarce knowledge about the status of wild animals, high superstition and myths associated with traditions that cause harm to wild animals. Hence, these activities of local people may lead the extinction of wildlife. Therefore, to protect the wild animals, the local traditional people should aware about the alternative method of treatment systems such as the use of medicinal plants instead of animal products. Study of Jaroli et al. [48] found that among 24 identified animals used by Garasiya people of adjoining areas of Mount Abu Wildlife Sanctuary, India, 16 animals (including elephant, tiger, sambar, Himalayan black bear) included in IUCN red list. If the people didn't think about the alternative methods of treatment systems, it will bring great problems on wildlife conservation. The global biodiversity crisis, caused mainly by anthropogenic actions, people overexploited the wild animals for ethnomedicine and created obstacles in animal conservation [38]. A similar type of problem was also reported in Brazil [42].
Extinction risk is very high for the vertebrates as compared to invertebrates [50]. Vertebrates are more prone to habitat loss, exploitation, poaching and illegal trade. Besides other studies, ethnomedicinal uses of vertebrates offer the reasons for increasing threats to their conservation. Therefore, this study is mainly devoted to vertebrates and not invertebrates. The ethnic communities and local healers need search for other treatment options such as plants instead of animals. For example, fruits of Rhus javanica can use instead of cooked blood of Golden jackal (Canis aureus) which was used treating asthma [24,51]. Seed oil of Impatiens scabrida can help to relieve body pain instead of fat of leopard (Panthera pardus) [24,52,59]. Similarly, powder prepared from the roots of Heracleum wallichii used treating stomach problems to substitute meat of Himalayan goral (Naemorhedus goral), Indian crested porcupine (Hystrix indica) and Liebig's frog (Nanorana liebigii) [53,54]. This study suggests that there is enough space for the researcher to document alternatives of the vertebrates for ethno-medicinal value.

Conclusions
The study is the first effort to document primary data of the ethno-medicinal knowledge about the use of vertebrates by the local people of Chitwan-Annapurna Landscape. Ethno-medicinal knowledge about vertebrates and their body parts and products play the vital role in conservation and consumption of those species. A total of 58 species of vertebrates used for the treatment of 62 human ailments which were grouped into 11 categories. Mammals contributed the highest number among them (n=24) in ethno-medicine. This study also indicated that more than 76% of vertebrate species were found to be used for the treatment of more than one ailment. The most commonly used species was Felis chaus (UV = 0.25) with 3 use-reports by 10 informants. Cardiovascular and dental problems had the highest ICF value (0.974) with cardiovascular problems having 351 use-reports for 10 animal species and dental problems having 77 use-reports for 3 animal species. The least ICF was found in ophthalmological problems (ICF=0.833, use reports=7 for 2 species). Traditional knowledge was more common among elderly people than young people because of long experiences of utilizing nature while lack of more knowledge among youth. Therefore, our study concluded that there is

Funding
The project was supported by the Nepal Academy of Science and Technology, Kathmandu and Institute of Botany, Czech Academy of Sciences, institutional support RVO 67985939.

Availability of data and material
All data collected and analyzed during the study are included in Table and figures of this manuscript.

Ethics approval and consent to participate
Permissions were taken from concerned authorities of the study area such as Department of National Parks and Wildlife Conservation, Division Forest Offices of Chitwan, Tanahun and Kaski districts and local authorities. Prior oral informed consent was obtained from the local people who participated in the interviews and surveys.     Table 4 Categories of ailments and informant consensus factor (ICF) for these categories.     Mode of preparation of medicine from body parts of vertebrates.