3.1.4. Thermal Ease

The natural habitat of the Punjab urial in Pakistan ranges from the dry, hot, and arid rocky mountains, to the high elevations in northern areas, which have extreme climatic conditions [29,30]. In captivity, there is a possibility of exposure for a long time to temperatures di fferent from those of the natural habitat, which can eventually result in heatstroke and stress in animals [10]. In order to assess thermal ease, two indicators were developed: "shelter availability" (4.1) and "shade availability" (4.2). These indicators were assessed by checking whether all animals could simultaneously access shade during hot weather and shelter when climatic conditions are not favorable. These indicators were already validated in [10,19] for dorcas gazelles and domestic sheep.

#### 3.1.5. Easiness in Movement

Wild ungulates usually roam over vast areas, and their home ranges may exceed several square kilometers [31]. Wild animals kept in relatively small enclosures are more prone to develop di fferent negative behavioral and physiological changes. All these negative developments are clear indicators of poor animal welfare [32]. According to [33], each Punjab urial should have a minimum area of 46.45 m2, and for every additional animal, this area should be increased by 25% (11.6 m2). Two indicators were developed in the current protocol, which had already been used and validated in [10,11,19]. The indicators included "total area of enclosure" (5.1), and "space (m2) o ffered per animal" (5.2), with the latter derived as the total enclosure area divided by the total number of animals in the area.

## 3.1.6. Standard Enclosures

The enclosures designed and constructed for breeding and conservation of wild animals should fulfill maximum criteria for standard enclosures. These criteria include fence structure and fixation to prevent the entry of other animals, especially small mammals. The fence should be covered so that animals do not have any visual contact with the outside environment if the aim is reintroduction. Each enclosure must have quarantine facilities, so that newly arrived animals can be held in isolation for observation, and sick or infected animals can be separated from other animals. Keeping in view the recommendations made in [34,35], four indicators, i.e., "fence structure" (6.1), "fence substratum" (6.2), "availability of quarantine" (6.3), and "number of quarantines" (6.4), were developed to assess enclosure standards.

#### 3.1.7. Lack of Injuries

Due to limited space and the presence of aggressive animals, especially when di fferent species are kept together, there is a chance of injuries [36]. Hoof injuries are common in artiodactylids [37]. There is a high chance of integument deformities and skin damage in captive animals. These deformities can result from any infectious disease, physical environment, aggression, or improper capture and handling of animals [10]. Two indicators were developed to assess lack of injuries: "integument deformities" (7.1) and "lameness" (7.2).

Integument deformities were assessed from close observation of the skin of animals. Hairless or damaged patches of skin of more than 2 cm were counted [10]. Animals were also observed for intraspecific aggression marks and injury marks from capture. Animals with "integument deformities" (7.1) were scored as follows: animals having several small lesions or a single lesion bigger than 2 cm<sup>2</sup> and hairless patches were considered to have "severe integument deformities"; animals having no lesions, but having hairless patches were considered to have "moderate integument deformities"; and animals having no lesions or hairless patches were considered to be normal with "no integument deformities".

All animals were observed for "lameness" (7.2) while they were moving. Animals having an apparent abnormality in walking or running were scored as "lame", while those having no apparent abnormality were scored as "not lame". The above indicators were already validated in [10,11,19].

#### 3.1.8. Lack of Disease

Common diseases found in Punjab urial (*Ovis vignei punjabiensis*) are viral infections, such as contagious ecthyma, and foot and mouth diseases (FMD). The major bacterial disease is pneumonia caused by *Pasteurella* spp. [17]. Other disorders that can a ffect the captive animals are behavioral disorders, birth problems, gastrointestinal infections, and trauma. Trauma mostly occurs due to intraspecific aggression, during capture and handling, or due to accident [10]. According to the American Association of Zoo Veterinarians [38], every facility holding wild animals, especially for conservation and breeding purposes, must have qualified veterinarians, a veterinary facility where sick animals can be treated, and a facility fornecropsy.

Six indicators were developed to assess the lack of diseases, including respiratory and gastrointestinal diseases, and health facilities. These indicators included "ophthalmic discharge" (8.1), "nasal discharge" (8.2), "labored breathing" (8.3), "diarrhea" (8.4), "availability of veterinarian" (8.5), and "availability of veterinary facility" (8.6). If any indicators among these were observed, animals were scored as "obvious", and animals having no sign of these indicators were scored as "non-obvious". All these indicators were validated in [25,27,39,40] for the welfare assessment of cattle.

#### 3.1.9. Displaying Social Behavior

Agonistic behavior is displayed by animals usually in response to stress, intraspecific and interspecific competition for resources, and mating competition [41]. This behavior can result in severe physical damage. *Ovis vignei punjabiensis* is a gregarious ungulate with multiple agonistic and a ffinitive behaviors [22]. In captivity, no studies have been conducted on these animals to evaluate their social behavior. Extreme and repeated agonistic behavior displayed by animals in captivity can be a sign of poor managemen<sup>t</sup> and welfare [10]. On the contrary, frequently occurring a ffinitive behavior represents satisfaction of animals concerning their environment, and hence good animal welfare.

Two indicators were developed to assess the social behavior: "a ffinitive interactions" (9.1) and "agonistic interactions" (9.2). These indicators were also validated in [10,21,25]. An ethogram (Table 2) was developed based on the social behavior of Punjab urial [22], and information was obtained from a short reconnaissance survey for this study.


**Table 2.** List of social behaviors included in the welfare protocol for Punjab urial.

> AFI = Affinitive interactions, AGI = Agonistic interactions.

#### 3.1.10. Group Dynamics

Punjab urial are gregarious ungulates [30] with different types of herds. Rams usually prefer to join herds in the rutting season; thus, the herd composition changes with the season. Three types of herds have been observed in wild Punjab urial: female herds exclusively comprising ewes and young; mixed herds containing one or more rams, ewes, and young; and male herds containing only males [22]. Husbandry guidelines sugges<sup>t</sup> that in reproductive groups of captive ungulates, one adult male should be kept with 3–7 females and their young, while 3–7 adult males should be kept in bachelor groups [10]. In order to prevent inbreeding, reproductive males should be replaced or exchanged after every two years and females after three years [34].

Following [10], two indicators—"herd size" (10.1) and "herd composition" (10.2)—were developed to assess the group dynamics. Wildlife enclosures and zoos have a current tendency to build larger and more naturalistic facilities. Sometimes it is encouraged to mix species with other sympatric species where they share the same space and resources, which may serve as a source of enrichment, especially for animals raised for conservation and reintroduction purposes [41]. The current protocol included another indicator "number of animals (other species)" (10.3) [10], to record the number of individuals of other species. In addition to the number, any information about interspecific interactions based on the behavior should be recorded, which could be very useful for establishing managemen<sup>t</sup> guidelines.

#### 3.1.11. Display of Other Behavior

Behavior that is invariable and repetitive without any apparent purpose is termed stereotypic behavior. Most of this behavior is due to stress, frustration, repeated attempts to cope, or failures of the central nervous system [27,42]. Sometime this behavior may help animals cope with hostile and difficult environments, but stereotypic behavior is generally regarded as an indicator of poor welfare [43]. There is no such information about stereotypic behavior in captive Punjab urial. Wild animals, especially ungulates in captivity, are more prone to develop oral stereotypies [44]. This protocol suggested that all animals should be checked for the presence or absence of "stereotypic behavior" (11.1). Animals should be scored as "stereotypes present" and "stereotypies absent" in the case of presence or absence of stereotypic behavior, respectively. Any behavior that is observed and recorded should be described properly [45]. The use of this indicator was strongly recommended in the welfare assessment captive animals and was validated successfully by [10].

Captive breeding programs can be promising for species that are raised for reintroduction, if environmental enrichment programs are encouraged. *Ovis vignei punjabiensis* prefers to live in rocky mountain areas with shrubs and grasses [29]. Although there are no husbandry guidelines for this species, guidelines for other species recommend providing opportunities for animals to perform their natural behaviors of browsing, grazing, running away, and hiding. In order to fulfill these requirements, it is recommended to make use of structural components like rocks, vegetation, and uneven ground, and to modify feeding techniques periodically with accompanying simulations of sounds and smell [10]. These practices will help produce captive-born animals suitable for release into the wild for reintroduction purposes.

To assess "environmental enrichment programs" (11.2) [10], the animals' access to opportunities such as browsing and grazing should be recorded. In addition, it should be taken into account whether certain objects, such as poles or sticks of di fferent shapes and sizes, are used; if yes, then when and how they are used must be described.

#### 3.1.12. Good Human–Animal A ffiliations

For keeping captive populations healthy, it is recommended to minimize stress usually associated with veterinary procedures [37]. Arranging proper medical training programs under the supervision of experienced sta ff can help to reduce stress and injuries while handling and treating animals. In case no "medical training programs" (12.1) are arranged, then it is of utmost importance that the facility must hold advanced "capturing, handling, immobilization, and translocation" (12.2) systems that can ensure the minimization of stress, damage, or any loss of animals. These indicators were already validated for dorcas gazelles in [10].

#### *3.2. Application of Punjab Urial Welfare Assessment Protocol*

The following results were obtained after implementation of the proposed protocol.

#### 3.2.1. Lack of Prolonged Appetite

According to the results obtained for body conditions (1.1) after application of the protocol, all animals had normal body condition with the exception of one adult male at CU1 with excessive body condition. According to the life history record of this male, it was never observed to have mated during the rutting season.

#### 3.2.2. Lack of Prolonged Thirst

The assessment of water sources (2.1) was conducted for all the three groups. At CU1, a concrete pond (0.91 × 0.91 m with a depth of 0.45 m) was present; CU2 had a concrete pond (1.52 × 1.52 m with a depth of 0.45 m) plus additional clay buckets (0.609 × 0.609 m); and CU3 had a concrete pond (2.74 × 2.74 m with a depth of 0.45 m) and three additional clay buckets (0.609 × 0.609 m). Water availability (2.2) and cleanliness of water sources (2.3) needed to be improved in CU2 and CU3. Both facilities had concrete ponds, but they were dry and the water in the clay buckets was not fresh and clean. Only the pond at CU1 had a su fficient supply of fresh water from a nearby spring. The study was conducted in the hot month of August and animals were frequently observed drinking water from this pond.

#### 3.2.3. Lack of Minerals

Salt licks were available (3.1) in sufficient quantity in each of the three facilities, and no animal was found to be licking objects (3.2). According to these results, animals showed no signs of mineral deficiency.

## 3.2.4. Thermal Ease

Each of the three facilities provided shelters (4.1) for animals. At CU1, there were three shelters in total: two of 3.04 × 13.04 m (metal) and one of 3.96 × 3.04 m with a wooden roof. All animals were provided with enough shade (4.2) during the hot summer. In addition to the shelters, CU1 had many large sheesham (*Dalbergia sissoo*) trees providing thick cover for the animals. Animals were observed preferring to stay in the shade of trees rather than in artificial shelters. One shelter (15.24 × 6.09 m) was provided at CU2, and two shelters (15.24 × 6.09 m each) were provided at CU3. In addition, enclosures held enough vegetation (*Acacia modesta*) to provide shade for the animals. Every animal had access to shade and shelter in bad weather conditions (hot weather and rainy days).

#### 3.2.5. Easiness in Movement

The total area of the enclosures (5.1) and the space offered per animal (5.2) varied across the enclosures. Being constructed in or on the boundaries of wildlife parks, every enclosure encompassed a large area, with sufficient natural vegetation and uneven ground. CU1 was the only enclosure which lay close to local settlements and roads (Table 3).

**Table 3.** Total enclosure area and area offered per animal in each herd, including individuals from other species (having the same approximate space requirements as Punjab urial).


a Punjab urial, *n* = 8; chinkara, *n* = 13; mouflon sheep, *n* = 3.

Although enough area was provided, sub enclosures should be designed and constructed within the main enclosures to separate different reproductive groups.

## 3.2.6. Standard Enclosures

All three enclosures were assessed for existing fence conditions (6.1): whether it was covered or not, or broken. The fences in all the three enclosures were single layered and uncovered, but not broken at any point. The location of the enclosures and thick vegetation prevented the animals from eye contact with the public. In CU1, due to comparatively less vegetation and nearby road and settlements, the fence should be double wired and covered in order to avoid any physical or frequent eye contact with the public.

Fence substrata (6.2) were assessed and found to be in good condition, thus preventing the entry of other small mammals into the enclosures. None of the three enclosures had quarantine facilities (6.3 and 6.4). For robust captive breeding and management, the availability of quarantine facilities should be ensured.

#### 3.2.7. Lack of Injuries

Animals were observed visually (also included the use of binoculars) for the assessment of integument deformities (7.1). No animal was found with any integument deformity. All animals were carefully assessed for lameness (7.2), but all the animals were normal.

#### 3.2.8. Lack of Diseases

Eight individuals (one adult male, three adult females, and four juveniles) at CU1 were observed to have ophthalmic discharge (8.1). None of the animals from CU1, CU2, or CU3 were observed to have signs of nasal discharge (8.2), labored breathing (8.3), or diarrhea (8.4). No classified veterinarian (8.5) or veterinary facility (8.6) was present at any of the three facilities. Veterinary records of each facility revealed that animal health checks were performed once a month, and the animals were checked by a classified veterinarian in case of mass infections. Keeping in view the budget constraints, optimum veterinary facilities in all the centers should be improved accordingly.

#### 3.2.9. Displaying Social Behavior

Results for a ffinitive interactions (9.1) and agonistic interactions (9.2) showed that the highest aggression was shown at CU3 (66.59%), followed by CU1 (43.64%) and CU2 (37.75%) (Figure 1). The results obtained from the Shapiro–Wilk test (W = 0.634, *p* < 0.05) were significant. Thus, our data was not normally distributed. The Kruskal–Wallis H test showed that there was a statistically significant di fference in behavior among the three groups ( χ2 (2) = 10.073, *p* < 0.05), with a mean rank of 87.43 for CU1, 80.22 for CU2, and 109.00 for CU3. Animals at CU3 shared the space with other animals (Table 3).

**Figure 1.** Percentage of a ffinitive and agonistic interactions of the total number of interactions recorded for each herd of Punjab urial assessed (*n* = 37).

While assessing the social behavior (9.1 and 9.2), the Punjab urial at CU3 showed aggression (chasing and head jerking) toward the chinkara. No obvious aggression was observed from Punjab urial toward mouflon sheep. All three species were observed maintaining approximately a distance of 10–20 m from each other.

## 3.2.10. Group Dynamics

An assessment of the number of Punjab urial (10.1) in all the centers showed that only animals at CU3 were sharing space with other species, including chinkara (*Gazella bennettii*; *n* = 13) and mouflon sheep (*Ovis orientalis*; *n* = 3) (10.3). Results for assessing herd composition (10.3) were: CU1, *n* = 23 animals (adult male = 5, subadult male = 1, female = 6, young = 11); CU2, *n* = 6 (adult male = 3, adult female = 1, young = 2); and CU3, *n* = 8 (adult male = 4, adult female = 2, young = 2). Life history records of Punjab urial at each of the three facilities showed that no male had been replaced with other males from a di fferent population. This situation was alarming and could potentially lead to inbreeding depression and drastic downfall of the population. These results strongly suggested that the exchange of productive males and females between different populations is necessary to avoid inbreeding.

#### 3.2.11. Display of Other Behavior

While assessing animals for stereotypic behavior (11.1), only two adult females and one sub-adult male at CU1 were found to move back and forth repetitively next to a fence and bite the fence. Regarding environmental enrichment (11.2), all the centers provided natural opportunities to forage, in the form of thick and tall vegetation for browsing. Due to comparatively fewer shrubs and less low vegetation at CU1, formal enrichment in the form of vertical poles or sticks to offer food should be encouraged. This will increase complexity in the physical environment of the enclosures.

#### 3.2.12. Good Human–Animal Affiliations

Each of the herds assessed at the three different centers had never been a part of any medical program (12.1). Very poor results were recorded regarding capturing, handling, immobilization, and translocation techniques in all the three centers. Animal capture was mostly conducted by chasing animals and confining them to a corner of the enclosure. Veterinary and post-mortem reports revealed the expiry of seven chinkaras and three Punjab urials during capturing and translocation, respectively.
