مطالعه آناتومیک و تشخیص استخوان­‌ها و دندان‌­های جانوری کاوش سال 1397 محوطه قره‌تپه سگزآباد (عصر آهن 2 و 3) و ساخت نمونه سه بعدی

Introduction
Zooarchaeology, a branch of biology, investigates and analyzes animal remains at ancient sites. It seeks to determine the position and role of animal species in relation to human survival and, in other words, to determine the interaction between humans and their environment. Examining the animal bones remains and classifying them into different species enables us to identify past climatic conditions, animal patterns, diversity, population size, subsistence economy, type of diet, and so on. Generally, various animal species adapt to different biological and living conditions; for example, the climatic conditions of an area require specific species in that region (Albarella, 2017). Zooarchaeology closely connects with comparative anatomy, utilizing it to identify the species of bone samples found in ancient excavations.
Another thing investigated in Zooarchaeology involves the identification of immature animal bone samples by examining the growth plates of the animal. The absence of finalized anatomical features in these bones, particularly in small ruminants, complicates their differentiation (Sisson & Grossman, 1975). In addition, detecting abnormal signs such as welded fractures, osteoporosis, and malformations holds significant importance. These abnormalities are detected and examined by imaging techniques used in veterinary science, such as radiography and CT scans. These techniques help us to extract more information and make more accurate diagnoses (Thrall Widmer, 2018). Also, detecting marks such as cuts (butchery), burns, and cooking in bone samples gives valuable insights (Donlon et al., 2020).
The study of societies in different parts of the Iranian plateau during the first millennium BC reveals some cultural and economic relationships, especially during the Iron II and III Ages. One of the most important sites of this era is Segzabad hillock, about 7 km north of Segzabad City, Boin Zahra County, within the Qazvin Plain. Recent research shows this hill has witnessed three distinct periods: The Copper Age, the late Bronze Age, and the Iron Age (Dehpahlavan & Jahed, 2021).
Excavations in this area have been focused on the Iron Age II and III layers. The objectives were to understand the reasons for the stagnation and end of the life of Qareh Tepe Segzabad, as well as to assess and recognize the cultural pattern during these periods. One of the essential findings of these excavations pertains to animal remains obtained from the residential, cemetery, and sedimentary layers. Based on the architectural spaces, pottery, ornaments, cylinder seals, weapons, and radiocarbon 14 results, the archaeological findings belong to the Iron II and Iron III periods (550-850 BC) (Fāzeli Nashli et al., 2011).
In the excavation project at Qareh Tepe of Segzabad, many questions are raised regarding the inhabitants’ economic and cultural interactions with their neighboring sites during the concurrent presence of cultural areas in the north-central plateau and the northwest and west districts of Iran in the Iron Age II and III. Undoubtedly, a detailed examination of the animal remains found from the excavation of the Segzabad hillock can answer most of these questions.
One of the archaeological discoveries in Qareh Tepe of Segzabad pertains to cylinder seals found by Dehpahlavan and Alinezhad (2022). Their study posits that these cylinder seals can be evidence of administrative signs and applications. These cylinder seals were used in decorative necklaces and probably had a religious use in their period.
Trebicka et al. investigated the remains of human bones in the excavations of Qareh Tepe of Segzabad. Their research mentioned the low frequency of caries, as low as 4%. Based on the observed evidence and the high percentage of injuries caused by trauma to the bones, they concluded that the level of interpersonal violence was high (Trebicka et al., 2019).
In another exciting discovery, Dehpahlavan & Jahed, (2021) found two tablet-like objects in this ancient site in their archaeological excavations and stated that the objects were used as counting games or game boards.
In addition to the mentioned instances, some anatomical studies have been conducted to help identify and separate the ancient bones of animals. In 2019, Chuang and Bonhomme conducted a study on the samples of domestic equids teeth discovered to distinguish the different species of this group of mammals. One of the things they mentioned is that mandibular molar teeth lack anatomical features that can be used to distinguish between species with high accuracy. Hanot and Bochaton (2018) conducted another study to separate donkey and horse bones. They pointed out their identification keys in their research and claimed that many bones of domestic equids could be identified, and their proportions could be determined within a collection. Salvagno and Albarella (2017) proposed a morphometric system for detecting and analyzing the samples. They noted that the efficiency of the new method will also be tested with discriminant analysis, providing an additional line of analysis to use in conjunction with other methods.
Despite the abundance of animal bones found in various ancient excavations, many of these excavated bones have not been studied and analyzed due to the lack of archeology experts in the country. As mentioned, a close relationship exists between the Comparative Anatomy of bones and Zooarchaeology. For this reason, this study was jointly designed and implemented by veterinary anatomy and archaeology experts. It should be noted that this form of cooperation between veterinary anatomy specialists and archeology collections has been undertaken before (Radmehr et al., 2003). Various studies have been carried out in the field of bone anatomy to identify and separate the bones of different species of animals (Shateri Amiri et al., 2020; Fatahian Dehkordi et al., 2022; Alizadeh et al., 2017; Yousefi, 2016).
In this study, by examining and typifying the remains of animal bones and teeth in the site of Qareh Tepe, Segzabad, and comparing them with the remains of previous eras, we try to explain and correctly analyze the subsistence economy of the site and answer the questions that arise in this context.

Materials and Methods

Bone and tooth samples
This study examined 1110 bone and teeth samples derived from the 2017 excavation in Segezabad hillock. First, each sample was washed with water, and after drying, primary classification and coding were done and placed separately in special zip kip packages. In addition to coding each sample, the required information was recorded on a sheet of paper and inserted into the corresponding package. The samples were transferred to the Osteology Lab of the Anatomy Department of the Faculty of Veterinary Medicine of the University of Tehran.

Anatomical study
Anatomical studies were done in several stages based on the samples. In the first stage, each specimen was assigned to its respective bone. Then, based on visible signs and structures, the taxonomic order to which the bone belonged in the animal classification was diagnosed. At this stage, the skeletal anatomy atlases available in the osteology laboratory and the collection of bones available in Anatomy Hall were used.
In the initial stage of sample separation, the samples were examined for the preliminary diagnosis of animal classification based on the information in “Sisson and Grossman’s the anatomy of the domestic animals” book (Sisson & Grossman, 1975). During this stage, the bones and teeth were classified into the following animal groups: Large ruminants, small ruminants, odd-toed ungulates (Perissodactyla), boars, camels, carnivores, rodents, and birds. In the second stage of separation, taxonomic division to species identification was done based on the available anatomical sources that will be referred to. The taxonomic division of this stage is detailed below. In the results section of the article, the number of diagnosed samples in each group will be mentioned.
Among the samples of adult horse bones, despite their distinctive anatomical features from donkeys, some cases were so small that they were identified as Caspian horses (a small horse breed native to Northern Iran). As a reference, we used the Caspian horse skeleton available in the Anatomy Department of the Faculty of Veterinary Medicine of the University of Tehran (Figure 1). It should be noted that the Caspian horse samples with three phalanges (the first, second, and third phalanges of a horse) were related to abnormal symptoms related to some diseases. To complete the diagnosis of abnormal cases, radiography was performed, the description of which is given in the relevant section.

In addition to the mentioned references, “Human and Nonhuman Bone Identification: A color Atlas” was also used to identify different species of animals (France, 2008). Regarding the Rodentia order, we should mention that the investigation was done to separate the rodent samples from the Lagomorpha order. But no sample of Lagomorpha order was found. Frequently, the cases of this order are mistakenly considered as rodents. One of the sources used for species separation of Aves Class samples was the “A Manual for the Identification of Bird Bones From Archaeological Sites” book (Cohen & Serjeantson, 1996). To identify tooth samples better, we used the “Cambridge Manuals in Archaeology: Teeth” book (Hillson, 2005). Finally, to examine the anatomical abnormalities of the bones, which indicated some problems and diseases, we used the book “Palaeopathology, Cambridge Manuals in Archaeology” for better examination and conclusions (Waldron, 2009).
Table 1 presents some of the most essential sources of calcification of all types of samples, including bones and teeth.

Radiography findings 
Radiographic images were prepared to complete the information obtained from examining some bone samples with abnormal symptoms. We used Toshiba, DC-12M radiography machine, and Carestream, Directview, Classic CR machine for this study stage. 
Specifically, to examine the first, second, and third phalanges of the Caspian horse, which belonged to the same horse, a series of radiographs were prepared using the following projection modes: Lateral, lateral oblique (dorsolateral-palmaro-medial oblique), medial oblique (dorsomedial-palmaro-lateral oblique), dorsopalmar and upright pedal route.
Radiography was performed with horizontal rays and technical factors set at mAs4 and Kvp45.

3D modeling
The bones with minimal damage or special characteristics were selected in this stage. The samples were transferred to the Small Animal Radiology Department Hospital of the Faculty of Veterinary Medicine of the University of Tehran, where CT scans were conducted.
The Siemens Somatom Spirit II CT-Scan machine was employed to prepare images. The technical parameters for this imaging protocol were as follows: Rotation time, l s; slice thickness, 1 mm; reconstruction interval, 0.5-1 mm; pitch, 1; x-ray tube potential, 120 kV; and x-ray tube current, 130 mA.
To facilitate a comprehensive examination, appropriate Window width and Window level settings were selected for each section graph within the CT-scan image results. Bone windows were used to check the images. 
For 3D modeling, first, the CT scan output of each sample was transferred to 3D Slicer 4.8.0 software in DICOM format to obtain a 3D sample with the .OBJ format featuring the bone pattern. Then, the .OBJ format file was transferred to the 3D Max 2018 software, where the Slicer software did the process of cleaning the wrongly detected items. The final editing was done in the 3D Coat 4.8.15 software, culminating in the 3D printing (Zehtabvar, 2022).

Statistical analysis
The collected data were entered into Microsoft Excel version 2013. Absolute frequency and percentage of relative frequency were used to describe the data.

Results
The specified orders in this study are as follows: Perissodactyla (Family: Equidae), Artiodactyla (Suborders: Ruminantia, Suidae, and Camelidae), Carnivora, Aves Class (Order: Galliformes) and Rodentia.
Regarding the order Perissodactyla, we should mention that the study samples were related to horses (Equus ferus caballus), and no donkey samples (Equus asinus) or Persian onager samples (Equus hemionus onager) were found among the samples. For this reason, the word “horse” has been used in the article’s tables, figures, and text for the samples identified in this group.
Regarding the Artiodactyla order, we should mention that from the suborder Ruminantia in this study, the samples related to cattle (Bos taurus), sheep or domestic sheep (Ovis aries), goat or domestic goat (Capra hircus), Persian gazelle (Gazella subgutturosa), Asian water buffalo (Bubalus bubalis) were mentioned separately in the tables, figures, and the text. In addition, in the Artiodactyla order, Wild boar (Sus scrofa) from the Suidae family and one-humped camel (Camelus dromedarius) from the Camelidae family were also detected separately.
In the case of the Carnivora order, we should mention that from the Canidae family, samples related to the Canis genus were identified as domestic dog species (Canis familiaris). 
Regarding the Rodentia order, it is to be noted that due to the lack of comparative anatomical sources for this order, the separation of different species of rodents was not done.
Regarding the Aves Class, we should mention that the samples were from the Galliformes order. However, the separation to the extent of species separation showed that all the samples were identified as chicken (Gallus domesticus).
In Tables 2 and 3, the absolute frequency and percentage of the relative frequency of animal bone samples from the exploration of Qareh Tape Sagzabad in 2018 were separately determined for each species. Then, the cumulative display of the samples diagnosed in each species is presented as total (Tables 2, 3)

Among the identified animal samples, the highest percentage of the relative frequency, with 30.09%, belonged to sheep samples. After that, samples of small ruminants with 26.94% could not be separated (goat, sheep, or Persian gazelle). After collecting small ruminants, the highest percentage of relative frequency, with 13.33%, was related to horses and domestic dogs, with 7.93%. After the domestic dog, the goats took 7.03%, then Rodentia at 4.68%. After Rodentia, cattle followed with a relative frequency of 3.60%. The relative frequency of one-humped camel was 0.99%. The percentage of the relative frequency of gazelle, Asian water buffalo, wild boar, and chicken was minimal and less than 1%. 

In Table 4, the absolute and relative frequencies of immature animal bone samples from the exploration of Qareh Tape Sagzabad in 2018 are separated based on the detected bones in each species. Data are displayed for each species as a total of the detected samples. Among the detected immature animal samples, the highest percentage of the relative frequency, with 81.82%, pertained to small ruminant samples, followed by carnivore samples (most likely domestic dogs) with 9.92%. Immature samples of odd-toed ungulates (Perissodactyla) accounted for 6.61% of samples, and samples of large ruminants accounted for 1.65%. Apart from the mentioned species, no immature bones of other species were observed (Table 4). However, in the case of immature bones, the distinction between the species of ruminants was not very accurate. Therefore, in this Table, small ruminants, large ruminants, odd-toed ungulates (Perissodactyla), and carnivores are collectively presented.

Table 5 lists the absolute and relative frequencies of bones with traces of human intervention (such as butchery effects, etc.) from the exploration of Qareh Tape Sagzabad in 2018. They were displayed separately by the identified bones in each species and the total samples detected for each species (Table 5). Among the samples with traces of human intervention, the highest percentage of the relative frequency (41.53%) belonged to sheep samples, followed by small ruminants with 37.38%. Finally, there were goat samples with 7.03%, horse samples with 5.11%, and cattle samples with 2.56%. In other species, the effects of human intervention, such as butchery, etc., were not observed (Table 5).

In the following, we discuss representative samples with butchery marks. Among the samples with butchery marks, a small ruminant immature metatarsal bone had cut marks in the distal part of the body (Figure 2).

Among these samples, there was a left talus bone related to sheep, which had cuts on the distal surface of the talus (Figure 3).

A left-side immature humerus bone from the small ruminant, which had cut marks on the cranial surface of the distal part of the body and distal extremity, was also detected (Figure 4).

A cornual process sample o a Persian gazelle was identified, which was cut into two pieces (Figure 5).

A left-side immature metacarpal bone sample of a small ruminant was found with cuts and scratches on the cranial surface of the body and signs of being chewed by carnivore teeth (Figure 6).

Another small ruminant immature metatarsal bone sample was identified with cuts and scratches on the cranial surface of the body (Figure 7).

Moreover, a sample of a small ruminant’s right immature metacarpal bone was found to have cuts and scratches on the cranial surface of the body, as well as signs of being chewed by carnivore teeth (Figure 8).

Lastly, one talus bone and one left-side calcaneus bone of sheep were identified; these two bones belonged to the same animal. The talus bone on the dorsal surface and the calcaneus bone on the lateral surface of their distal part had cut marks (Figure 9).

Three phalanges (first to third) were identified among the samples related to a horse. Based on the structure and size of the bones, they were related to the limb of a horse. Although they were smaller than the usual norm of an adult horse, they did not belong to a donkey either. By matching with the existing bone samples from the Caspian horse, it was found that these three phalanges belonged to the forelimb of an adult Caspian horse (Figure 10).

As mentioned, abnormal cases related to the disease were observed in these three phalanges, which were then investigated through radiography. The observed symptoms included mild side bone of the P3 (lateral and medial), mild new bone formation (high ringbone) of dorsomedial and dorsolateral proximal P2, and mild osteoarthrosis of the pastern joint (Figure 11).

Also, among the horse samples, a right humerus bone sample with cutting marks on the caudal surface of the distal part was detected (Figure 12).

Additionally, Figure 13 shows the bones from which the 3D reconstruction was performed, and the 3D printed samples have been shown.

All the information mentioned in the tables about the number of different bones are displayed in Figures 14, 15 and 16.

Discussion
The collection of remains of animal bones from Qareh Tape Sagzabad (Qazvin Plain, Iran) in 2018 encompasses 11 species. Small ruminants had the highest percentage, and then horse samples. The existence of this number of small ruminants, which need a suitable environment for grazing and feeding, indicates a suitable habitat for their breeding and feeding (Radmehr, 2012). Talai, (1983) have previously mentioned the favorable environmental conditions of this region. Therefore, in Qareh Tape Sagzabad in Iron Age II and III, small ruminants (sheep and goats) played an essential role in the subsistence economy.
One of the remarkable findings about the small ruminant samples separated at the species level in this study points to the higher number of sheep samples than goats. Goats can climb, stand on their hind legs, jump, and cross difficult areas to reach a safe area. Therefore, goat herds cover more distant areas than sheep when crossing pastures, and their control is more challenging (Zeder & Hesse, 2000). Goats have elongated limbs and thinner trunks than sheep. Such features have made the goat suffer less damage than the sheep in times of danger and sress. Goats can cleverly flee when faced with an unexpected threat, while sheep generally flee with their flock. Comparative analyses of feed consumption between goat and sheep reveal that goats have higher feed consumption. This difference is due to their higher net energy requirements for maintenance. Strong evidence supports that goats require more energy for maintenance than sheep, and goats’ metabolic requirements per kilogram of body weight are more similar to those of cows. Therefore, it makes sense for goats to consume more food than sheep to maintain body weight (Pugh & Baird 2012; Zeder & Hesse, 2000). Based on these facts, the conditions for keeping sheep seemed more suitable.
Since a small percentage of small ruminant bone samples were immature and the ossification centers of many bone samples were closed, they must be kept for a long time without being butchered to use their meat. Therefore, their primary reason for keeping them was to use their milk and wool in addition to meat. The same situation is observed for large ruminants, which may have been used for other purposes, such as carrying loads and working in the field (Reitz & Wing 2008).
In Qareh Tape Sagzabad, during Iron Age II and III, the number of odd-toed ungulates (Perissodactyla), especially horses, shows a significant increase compared to the Neolithic periods and the beginning of the complexity of Iron Age I technology. This shift was probably due to the use of horses and donkeys in agriculture and transportation (Talai, 1983).
Regarding the samples of Caspian horse phalanges, the observation of the abnormal cases and their radiographic confirmation suggests that side bone may result from factors such as hoof imbalance or limb imbalance as well as overweighting. It should also be mentioned about ringbone that this complication can be caused by repeated blunt trauma or repeated mild trauma. In the case of osteoarthrosis, it can also be caused by wear and tear (Thrall & Widmer, 2018). Considering the examined sample’s maturity and the mentioned complications, it is plausible to infer that the examined Caspian horse may have suffered these complications due to years of carrying and riding, and it was probably removed for this reason.
In the samples examined in this study, after horses, most samples belonged to domestic dogs. Judging from the frequency of this species’ samples, it seems that keeping domestic dogs with different purposes was common. Considering the abundance of examples pertaining to sheep that were kept in herds, using a domestic dog to protect the herd is an acceptable scenario. In addition, the bones that had cut marks and the signs of being chewed by a carnivorous animal were observed, indicating the food prepared by humans for these domestic dogs.
In this study, after domestic dogs, the highest frequency was related to domestic cows. With a lower percentage than in previous cultural periods (Neolithic and Iron Age), as well as the lack of remains of immature bones and butchery in this animal species, it is more logical to infer that this species was in the service of humans in farms and agriculture and not a source of animal protein. In addition, the presence of an Asian water buffalo hand bone indicates the possibility of wild ruminants in this area. Of course, we should note that in several parts of Iran, such as Azerbaijan, Mazandaran, and Khuzestan provinces, Asian water buffalo are domesticated and bred for various purposes (Nasserian & Saremi, 2007; Zhang et al., 2020). Considering the small number of bone samples of Asian water buffalo in this collection, the presence of this animal is not necessarily related to the life of this species in this region; instead one sample might be brought to this region by humans either as a living animal or as bone remains.
Rodents are considered invasive animals in this area, burrowing in the layers of the earth, and in some situations, they were probably buried there (Reitz & Wing 2008).
In the identified samples of this study, the bones of Persian gazelle, wild boar, and chicken were minimal. It should be noted that none of these bones had cuts or butchery marks. The presence of these samples seems unrelated to food consumption (Reitz & Wing 2008).
According to Talai, the Segzabad settlement formed due to the migration of “newcomers” without roots in the region. Talai considered favorable environmental conditions the most important factor for this migration. The presence of bones related to the one-humped camel among the samples of the studied bones, often associated with long-distance travel, can confirm the hypothesis of the migration of newcomers to this region or the prevalence of long-term travel (Talai, 1983).
In this study, the samples of immature bones of small ruminants, large ruminants, and odd-toed ungulates (Perissodactyla) were most likely from horses. The samples of carnivores were most likely domestic dogs, and no other animals were found. The presence of these immature bones can indicate their domestication and cultivation (Reitz & Wing 2008). The highest number of immature bones detected was related to small ruminants, followed by domestic dogs and horses, and the lowest number belonged to large ruminants.
Another aspect explored in this study involved the 3D prototyping the identified bones. In similar studies, efforts have been made to create archives of 3D images of bones from Zooarchaeology studies. This study used CT scan technology to make samples, but in most similar studies, 3D laser scanning or 360-degree photographs were used. The advantage of using methods such as CT scanning is that the mentioned methods record only bone surface information. However, bone depth and surface information in CT scans can be used in further studies (Betts et al., 2011).
Qazvin Plain is considered a semi-arid region because the area’s annual rainfall fluctuates from 200 to 300 mm (Talai, 1983). This region is in the climatic division of the regions with severe semi-desert and cold, dry climates. In addition, in the statistics obtained in this article, species of animals such as goats and sheep that were domesticated show a higher percentage than other animal remains. Therefore, suitable conditions for keeping these animal species can be predicted considering the region’s climatic conditions.

Ethical Considerations

Compliance with ethical guidelines
The all experimental procedures were approved by the Local Ethics Committee of Faculty of Veterinary Medicine, University of Tehran (No.: 30704.6.4).

Funding
This work was supported by the Faculty of Veterinary Medicine, University of Tehran.

Authors' contributions
Conceptualisation: Omid Zehtabvar; Methodology, data curation and resources: All authors; Formal analysis, funding acquisition, investigation, project administration, software, supervision, validation, visualisation, writing – original draft, review & editing: Omid Zehtabvar, Mostafa Dehpahlavan and Hesameddin Akbarein.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
The authors express their appreciation to everyone who assisted us in this study.

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