**4. Discussion**

The current project was designed to develop, for the first time in Pakistan, a baseline protocol to assess the welfare of wild ungulates in captivity. The assessment of resources, management, and animal-based measures are collectively termed as animal welfare assessments. In order to have a clear idea about the welfare of a group of animals in captivity, it is difficult to achieve the goals by having a single indicator or very few indicators. Thus, to have a complete and appropriate welfare assessment for a particular species, it is important to have a combination of several indicators according to the biology and ecology of that species [21]. The protocol proposed in this study is based on the welfare protocol for domestic sheep. This protocol differs from the domestic sheep welfare protocol in terms of the number of indicators, consisting of an extended list of 31 different animal- and resource-based indicators. In this newly developed protocol, we found that some indicators are difficult to assess with accurate results, especially in wild animals, when they are kept in large enclosures with dense vegetation. Although we used binoculars to assess integument and skin deformities (indicator 7.1), it was difficult to determine if there were any small lesions or patches on the body. According to [46], there is a high possibility of high levels of aggression and fights in wild ungulates in captivity. Detailed observations of skin and other integuments are thus very important. We do not encourage excluding this indicator; rather, we sugges<sup>t</sup> the use of more powerful binoculars or a high-resolution camera to obtain clear pictures of the animals.

Our protocol is based on the welfare protocol for domestic sheep, but we excluded the criteria 'positive emotional state'. There is a lack of information relative to this subject in captive ungulates [10]. This state includes pleasure, comfort, confidence, and interest. The aim of animal welfare assessment is to determine positive emotional states, or reduce undesirable experiences and increase opportunities

for animals to have more healthy and positive states [47]. We sugges<sup>t</sup> that these criteria should be included in developing welfare assessment protocols for any wild species in captivity.

An emerging trend in establishing zoos and enclosures is to provide large and more natural environments for captive animals [48]. The current protocol found the area requirements for captive Punjab urial very true-to-life and acceptable in each of the three enclosures examined. All three enclosures offered vast areas to the animals, with natural habitats utilizing natural vegetation and uneven ground. All the animals could easily experience grazing, browsing, and athletic activities. With the exception of CU1, where animals were observed to be moving back and forth and the enclosure is located in close proximity to local settlements and roads, so stereotypic behavior was recorded during the application of the current protocol. According to [49], eye contact of visitors and wild animals in captivity can result in stress and stereotypic behavior. Following [34], fences at CUI should be covered with raffia in order to avoid frequent eye contact between the animals and the public.

Interspecific aggression has been mostly documented in carnivores [50], while such information for wild ungulates is scarce [51]. Interspecific aggression can possibly increase intraspecific aggression. In our study, we found that animals in CU3 showed the highest aggression (66.59%), followed by CU1 (43.64%) and CU2 (37.75%). We assumed that the higher aggression in CU3 was due to the presence of other species [52]. Punjab urial males were frequently observed chasing chinkara, showing comparatively less aggression toward mouflon sheep. We also recorded counter aggression from chinkara males toward Punjab urial and mouflon sheep. According to [36], interspecific aggression is usually greater between distantly related species than closely related species, and it is recommended to separate the species with aggressive males. Our results suggested isolating chinkara from Punjab urial. Regarding stereotypic behavior, it was observed only in three animals (two adult females and one subadult male) at CU1. This facility had less natural vegetation as compared to the other two facilities, where the animals did not show any stereotypic behavior. According to [45], captive ungulates have a high tendency to produce oral stereotypies when they have limited opportunities for natural foraging; those findings are in agreemen<sup>t</sup> with results produced from the current study.

Recently, medical training programs and training techniques have been practiced and understood in modern zoos and facilities. These methodologies are frequently used and applied in different species, including big cats, elephants, giraffes, and apes. There is a lack of implementation of such programs in ungulates [10]. We consider it important to add a medical training program (12.2) because, if properly practiced, it is a promising means of reducing the stress caused by veterinary techniques. For capture of animals, every facility must have the right material (capture enclosure, net, handling crush, and dart gun), coupled with an experienced team, in order to avoid trauma and other serious injuries [34]. During application of the proposed protocol, it was found that several animals had expired during capture and translocation. These results make the medical training program a top priority in Punjab urial and other associated captive ungulates.

The development of this welfare assessment protocol is a leading documented work in developing a scientific and standard tool for the measurement of welfare in Punjab urial *(Ovis vignei punjabiensis*). Using the Welfare Quality® protocol for farm animals as a reference, welfare assessment protocols for several wild species have been developed, including those for mink (*Neovison vison*), foxes (*Vulpes* spp.) [47], dorcas gazelles (*Gazella dorcas*) [10], and bottlenose dolphins (*Tursiops truncatus*) [47,53].

Welfare assessment protocols are developed with the aim of assessing the welfare level in captive animals, to discover limitations in captive breeding, and to ensure optimal welfare through recommendations. Protocols that are practical and easy to be applied are considered successful protocols. Protocols developed for different species differ in terms of time for their implementation. In the case of mink and foxes [47], the developed welfare assessment protocol needs three visits to each farm. In the case of bottlenose dolphins [53], the protocol requires two days for a complete welfare assessment of a dolphin pod including up to 10 individuals. Our protocol includes an extensive set of 31 indicators, and thus has some practical challenges. According to [10], protocols for dorcas

gazelles require less than 6 h per herd. During the application of the proposed protocol, the largest herd of Punjab urial (*n* = 23) was assessed at CU1, and all the indicators were assessed in 5 h per herd. Our protocol is in early-stage development for assessing the welfare in captive Punjab urial and endorsement is still needed; however, its application to the three di fferent herds and the results obtained allowed to identify some areas in all the facilities which need to be improved.
