Caspian Horse Research Articles

Anatomical Study and Determination of the Animal Bones and Teeth Samples of the Excavation of Qareh Tape Sagzabad (Qazvin Province, Iran) in 2018 (Iron Age II and III) and Making 3D Models

Background: Zooarchaeology shares close ties with Comparative Anatomy of Veterinary Medicine. This scientific discipline involves the study of the bones of various animals, using anatomical information to explore the place and role of these animal species in life and human survival, thereby enriching our knowledge about the interaction of humans and their ecosystems. Objectives: Using available anatomical information, this study investigated the typology of bone remains and animal teeth in the area of Qareh Tepe, Segzabad (Qazvin Province, Iran). Another goal was to detect the abnormal effects created by humans or animals in bones, such as cuts, burns, or chewing effects by carnivorous animals. By extracting this information, it is possible to analyze the livelihood economy of that historical site correctly. Methods: A total of 1110 bone and teeth samples excavated in 2017 were analyzed. The samples were transferred to the Osteology Lab of the Anatomy Department of the Faculty of Veterinary Medicine, University of Tehran, Tehran City, Iran. Anatomical studies were conducted in several stages based on the excavated samples. Results: Among the detected animal samples, the highest percentage (30.09%) was related to sheep samples, and then the samples of small ruminants (26.94%) (which could not be separated). The percentages of gazelle, Asian water buffalo, wild boar, and chicken were trivial and less than 1%. In this study, abnormal symptoms were detected in the phalanges of the Caspian horse. Conclusion: One of the remarkable points about the small ruminant samples separated at the species level in this study is that the number of sheep samples detected surpassed that of goats. It seems that the conditions for keeping sheep were more suitable. The number of Perissodactyla, especially horses, shows a significant increase compared to the Neolithic periods and the beginning of the complexity of Iron Age I technology. This finding indicates the use of horses and donkeys in agriculture and transportation activities.

Investigation of High-level Aminoglycoside Resistance in Enterococcus spp. Isolated from Caspian Horse Feces in Guilan Province, Northern Iran

Investigation of High-level Aminoglycoside Resistance in <i>Enterococcus spp.</i> Isolated from Caspian Horse Feces in Guilan Province, Northern Iran

Comparison of principal component analysis (PCA) and discriminant analysis of principal component (DAPC) methods for analysis of population structure in Akhal-Take, Arabian and Caspian horse breeds using genomic data

Comparison of principal component analysis (PCA) and discriminant analysis of principal component (DAPC) methods for analysis of population structure in Akhal-Take, Arabian and Caspian horse breeds using genomic data

Stan hodowli i użytkowania koni kaspijskich w Europie i na świecie

The Caspian horse is one of the oldest horse breeds in the world and probably the first oriental domesticated horse. For centuries thought the breed had been considered extinct, until it was "rediscovered" in 1965 in Iran. The breed is quite important in the history of horse breeding, however, it is still little known. The aim of the work was to present the characteristics of the breed (conformation, characteristics and usage) and to assess the current state of the population in Europe and around the World. A survey was conduct among 18 breeders and included 120 horses. The population size was estimated based on the analysis of two breed registries. This study confirms and supplies information about Caspian horses available in the literature. Caspian horses can be very useful in refining small breeds of horses and could bring many profits in Polish breeding of sport ponies.

تعیین مقدار قابل ارجاع برای اندازهگیری میزان اشک چشم با استفاده از نوار آزمایش اشکی (STT) در اسبچه خزر

BACKGROUND: The Schirmer tear test (STT) is widely used in both human and veterinary ophthalmology. Two types of STTs have been developed: STT I and STT II. OBJECTIVES: The purpose of this research was to determine the reference value for measuring tear production of the Caspian horse by using the Schirmer tear test strip. METHODS: Twenty apparently healthy male and female Caspian horses, aged between 2 and 15 years old and weighing between 192±32 Kg were selected. After physical examination and reviewing the horse's health history and assuring their health, the Schirmer tear test was performed. To produce a higher volume of tears in horses, compared to other animal species, we used the Schirmer tear strip (Schirmer‐ Eickemeyer®, Germany), for 30 seconds instead of one minute in the lower conjunctival sac. In this study, we investigated the effect of age and gender on tear production in the Caspian horse, as well as the comparison between the left and right eyes tear production. RESULTS: The study shows that the results of STT I in the study animals illustrated the mean±SD STT I value was 23.38±3.6 mm/min (range, 15–30 mm/30s). Based on the results of this study, the variants of age and gender had no significant effects on the STT1 results in the Caspian horses. No significant difference was noted between the amount of tear production in the left and right eyes. CONCLUSIONS: Considering the importance of rapid diagnosis of dry eye syndrome and preventing its subsequent disorders such as Keratoconjunctivitis Sicca, the results of this study can be used as the reference value in the future to measure the amount of moisture of caspian horses of the Caspian horse.

Determination of the reference value for intra ocular pressure (IOP) in the Caspian miniature horse

Assessing the intraocular pressure is a key parameter for eye examinations used to diagnose diseases of this organ of the body. Determining the IOP requires a tool that is fast, accurate, portable, and functional. Currently, there are accepted methods for determining IOP, which include rebound tonometry and the Applentation method. This study aimed to determine the reference value for intraocular pressure by using rebound tonometry in the Caspian horse. In this regard, 20 apparently healthy Caspian miniature horses, both male and female aged between 2 and 14 years old and weighing between 150 and 200 kg were selected. The total mean and standard deviation of the results obtained from the study of intraocular pressure in 20 Caspian miniature horses were calculated as 32.9±6.7 mm/Hg. Gender had no significant effect on IOP (p >0.05). No significant differences were seen between the mean IOP values of the left eye compared to the right eye. Given the importance of early diagnosis of glaucoma and the prevention of subsequent disorders, it seems that the obtained results could be used as reference to measure the intraocular pressure of Caspian miniature horses.

Genetic Diversity of Persian Arabian Horses and Their Relationship to Other Native Iranian Horse Breeds.

The principal aims of this study were to explore genetic diversity and genome-wide selection signatures in Persian Arabian horses and to determine genetic relationship of Persian Arabians with other Iranian horse breeds. We evaluated 71 horses from 8 matrilineal strains tracing to 47 mares from the mid to late 19th century, using the equine 670k single nucleotide polymorphism (SNP) BeadChip. Mean observed and expected heterozygosity were (0.43) and (0.45), respectively, average inbreeding measures (inbreeding estimates based on runs of homozygosity and pedigree information) were low, indicating high genetic diversity in Persian Arabian horses. Analysis of population genetic structure using STRUCTURE and principal component analysis suggested that Persian Arabian horses can be divided into 3 groups, however the groups do not match traditional matrilineal strains. In total, 15 genomic regions were identified by at least 2 of the 3 implemented methods, Tajima's D, H, and H12, as potentially under selection in Persian Arabian horses. Most of these peaks were found on chromosome 9, overlapping with QTLs previously associated with horse temperament. Biological function analysis of identified candidate genes highlighted enrichment of GO term "response to lipopolysaccharide" and KEGG pathway "chemokine-mediated signaling pathway," which are associated with immune responses and may have been targets of selection in Persian Arabian horses. Independent analyses of SNP data from 30 horses of 4 other Iranian breeds suggested distinct population structure between Persian Arabian, and Turkemen and Caspian horse breeds. Overall, the results of this study suggest a rich genetic diversity in the Persian Arabian horses and a clear genetic differentiation with Turkemen and Caspian breeds.

A Nonsynonymous Change in Adhesion G Protein–Coupled Receptor L3 Associated With Risk for Equine Degenerative Myeloencephalopathy in the Caspian Horse

Equine degenerative myeloencephalopathy (EDM), a neurological disease of young horses, causes progressive development of symmetric ataxia predominantly in the pelvic limbs. Equine degenerative myeloencephalopathy is likely inherited and with no known treatment affected horses frequently need euthanasia. Alpha-tocopherol deficiency during early life appears to contribute to the phenotype. This study sought to identify any genetic variants correlated with EDM in Caspian foals. Two half-sibling EDM-diagnosed cases were genotyped at 52,063 loci and evaluated by the Autozygosity by Difference statistic. Additional horses not affected by EDM were used for genetic comparison to identify regions unique to the case phenotype. The associated region on chromosome 3 contains only one gene encoding adhesion G protein–coupled receptor L3 (ADGRL3). Adhesion G protein–coupled receptor L3 is a member of the latrophilin subfamily of G protein–coupled receptors and may contribute to attention deficit/hyperactivity disorder in humans and hyperactive motor function in mice and zebrafish. Analysis of the predicted coding regions for Equine ADGRL3 in affected horses revealed a nonsynonymous single nucleotide polymorphism at Chr3:71,917,591 bp. Caspian and Caspian cross-relatives (n = 81) of the two initial cases and unrelated horses from similar breeds (n = 130, including Arabians, American Miniatures, and Shetlands) possessed this allele at 5% frequency, with no homozygotes observed within the non-Caspian breeds. This study suggests that a polymorphism in ADGRL3 could contribute to a genetic predisposition to Caspian horse EDM.

Retraction: Genetic characterization and assessment of demographic bottleneck in Caspian horse population.

[This delete the article DOI: 10.14715/cmb/2017.63.11.16.].

Establishment and characterization of Caspian horse fibroblast cell bank in Iran.

Caspian horse, a rare horse breed found in 1965 by Louise Firouz in northern Iran, is a small horse which is reported to be in danger of extinction in its original homeland. There seems to be a great need to prevent extinction of this valuable horse. In this study, 51 fibroblast cell lines from Caspian horse ear marginal tissue were successfully established by sampling 60 horses using primary explant technique. Cells were authenticated and growth curve was plotted. According to results obtained, population doubling time (PDT) was calculated 23±0.5h for all cell lines. Multiplex polymerase chain reaction (multiplex PCR) revealed that cell lines had no cross-contamination with other species. Bacteria, fungi, and mycoplasma contamination were checked using standard methods such as PCR, direct culture, and Hoechst staining. In addition to providing a valuable source for genomic, postgenomic, and somatic cloning researches, the established cell lines would preserve Caspian horse genetic resources. It will also create an accessible database for researchers.

Effects of Dietary Thyme (Thymus vulgaris) and Fish Oil on Semen Quality of Miniature Caspian Horse

Thyme (Thymus vulgaris) is a subshrub from the lamiaceae family with plants that are rich in essential oils and antioxidative phenolic substances. The aim of the study was to investigate the effect of dietary thyme and fish oil supplementation on the semen characteristics of miniature Caspian horse. Sixteen stallions were randomly allocated into four groups and received four different diets: unsupplemented control diet, supplemented with fish oil at 2.5% dry matter intake (DMI), supplemented with fish oil (2.5% DMI), and thyme (0.02% DMI), and supplemented with thyme (0.02% DMI). All experimental diets were formulated according to National Research Council (1998). Semen was collected at 0, 30, 60, and 90 days. The semen samples were cooled and preserved at 5°C. Cooled diluted semen samples were evaluated in vitro by microscopic assessments of chilled sperm motility, acrosomal and other abnormalities (head, midpieces, and tail), viability (evaluated by Eosin–nigrosin), and plasma membrane integrity (evaluated by hypo osmolarity swelling test), and the level of malondialdehyde (MDA) was determined during cool storage 0, 24, and 48 hours after collection. The results showed that total and progressive sperm motility and plasma membrane integrity and functionality in all groups were significantly decreased with increasing storage time. On the other hand, the level of MDA in all groups was significantly increased with increasing storage time. Also, the results showed that most sperm quality parameters in this study were significantly higher in fish oil–thyme and fish oil group compared with thyme and control groups after 24 and 48 hours of storage at 5°C. We concluded that dietary supplementation of fish oil and thyme can improve sperm quality in miniature Caspian stallions during storage in cool condition via increasing total and progressive motility and plasma membrane integrity and functionality. More advances in vitro evaluations and artificial insemination are required to reveal the exact effects of thyme on miniature Caspian stallion sperm quality and its fertilizing ability.

Phenotypic analyses support investigations of phylogeny in the Skyrian pony and other breeds

Although genetic analyses suggest that the Skyrian pony is unrelated to other breeds, it shares some physical features with Exmoor ponies, thought to originate in the primitive pony referred to as Pony Type 1, and the Caspian horse, thought to be descended from Horse Type 4. To test the hypothesis that the Skyrian shares the Exmoor’s origin in Pony Type 1, comparisons were made of defined physical characters and morphometric measurements amongst Skyrian ponies, Exmoor ponies and Caspian horses. The average mean character differences were 0.56 ± 0.12 between the Skyrians and Exmoors, 0.43 ± 0.15 between the Skyrians and Caspians and 0.83 ± 0.06 between the Caspians and Exmoors. This approximates to reports comparing other horse breeds and breeds in other species, confirming that the Skyrian is a distinct breed. Using two-way analysis of variance (ANOVA), Friedman’s ANOVA and post hoc tests, no significant differences were found between Skyrians and Caspians in the eye, neck, body and limb shape (P = 1.000) nor shoulder angle (P = 0.222) nor in the ratios of body length:elbow height, neck length:neck circumference and heart girth: height to withers. However, there were significant differences ( P < 0.001) between Skyrians and Exmoors for these physical characters and ratios. While significant differences existed between Skyrians and Caspians in cephalic profile, parietal crest and tail placement (P < 0.001) and there were similarities between Skyrians and Exmoors in these physical characters (P = 1.000 or 0.098), overall the Skyrians appeared phenotypically closer to the Caspian, but unrelated to either breed. This investigation enabled the evaluation of a methodology comprising systematic, comprehensive investigations of phenotype, with data reduced and analysed by appropriate statistical methods, to clarify equine breed ancestry and phylogeny. The studies confirmed that the analysis of phenotype has utility and potential for investigations of phylogeny in species where there may be a paucity of information from the genetic base. Moreover, this approach has been shown to support and augment knowledge derived from studies based on genetic testing and provide a cost-effective and easily performed method of determining relatedness amongst equine breeds.

The Effects of Centrifuged Egg Yolk Used with INRA Plus Soybean Lecithin Extender on Semen Quality to Freeze Miniature Caspian Horse Semen

The aim of this study was to determine the synergistic effects of centrifuged egg yolk (EY) and soybean lecithin on post-thaw Caspian horse sperm motility, morphological abnormalities, and assessment of membrane integrity. The centrifuged EY (CEY) was added at concentrations of 2% and 4% to a defined INRA plus 1.25% soybean lecithin extender used to freeze Caspian horse semen. In this experiment, ejaculates collected from each Caspian horse (n = 4) were divided into three equal aliquots and diluted in CEY 2% (INRA2), 4% (INRA4) supplemented, and without any CEY (INRA0) in INRA plus 1.25% soybean lecithin extender, respectively. Thereafter, samples were frozen and thawed following a standard protocol. Sperm cryosurvival was evaluated in vitro by microscopy assessments of post-thaw sperm motility (by means of computer-assisted semen motility analysis [CASA]), acrosomal and other abnormalities (head, mid-pieces, and tail) and plasma membrane integrity (evaluated by HOST). In Caspian stallion, semen extended with INRA2 had significantly higher CASA motility and CASA progressive motility than those extended with the rest of extenders after freezing and thawing (P < .001). There was no significant difference in path velocity (VAP), VCL, and ALH among three groups (P > .05). For straight line velocity (P < .01) and LIN (P < .001), the highest values were obtained from the INRA4 group. The highest percentages of acrosomal and other abnormalities were found in semen diluted in INRA4 (P < .001). In the group frozen INRA2, the percentage of membrane integrity was significantly higher than that of the other groups (P < .001). The use of CEY 2% in combination with soybean lecithin significantly improved Caspian horse semen freezability.

Pregnancy in the Caspian Miniature Horse Using Frozen Semen Cryopreserved with the EquiPRO CryoGuard Freeze Medium and Customized Freezing Protocols

The primary goal of this project was to establish a protocol to freeze the sperm of the Caspian miniature horse in an attempt to start an intensive artificial insemination program to effectively increase the population of this breed, which has been listed as “Critical Rare Breed” by the American Livestock Breed Conservancy and is in danger of extinction. Commercially available equine freezing medium (EquiPRO CyoGuard Complete egg-yolk extender) was used for the initial setup of two different freeze protocols: slow and fast. The fast-freeze protocol had slightly better postthaw results and was used for a fertility demonstration. Five mares of proven fertility, aged 3 to 12 years, were used in the fertility trials, two of which resulted in pregnancy. This is the first report of pregnancy in the Caspian miniature horse using frozen semen, and the results seem to be a promising start to an extensive program to help this endangered breed, although further research on freezing protocols and conditions for this process are necessary to further improve the survival of semen and pregnancy rate.

Perineal sarcoid in a Caspian miniature horse

An 8-year-old stallion male Caspian miniature horse was acquired by the Veterinary Teaching Hospital, Islamic Azad University from a commercial source for teaching veterinary students. One year later, during an annual health screen, a mass was noted in the perineal region. The mass was surgically removed. Histologic examination revealed that the mass was a relatively well-demarcated, unencapsulated neoplasm composed of streams, bundles, and whorls of neoplastic spindle cells that had oval to fusiform nuclei with stippled chromatin, variably distinct nucleolus, and moderate amounts of eosinophilic cytoplasm. Based on the morphologic and histologic findings, a diagnosis of equine sarcoid was made. Results of bacteriologic tests and fungal culture of the mass were negative. By re-examining the horse 3 weeks after surgery, the affected site was completely healed. To the authors’ knowledge, this is the first report of sarcoid in a Caspian miniature horse.

Analysis of serum proteins electrophoresis in clinically healthy miniature Caspian horse

The Iranian miniature Caspian horse is the most ancient domestic breed of horse; it is the likely ancestor of all modern breeds of hot-blooded horses. Blood serum protein analysis has not been sufficiently studied in miniature Caspian horses. Since serum electrophoresis may be an important preclinical tool for diagnosis of equine diseases, evaluation of serum protein fractions in miniature Caspian horses using acetate cellulose strips was undertaken. In addition, serum protein concentrations were also examined. Blood samples were collected from 30 clinically healthy horses with and without anticoagulant. Using cellulose acetate electrophoresis (CAE), serum samples were fractionated and absolute and relative concentrations from each sample calculated. From 30 samples, 26 samples (86.6%) had five protein fractions and four samples (13.4%) had four fractions. Samples with five fractions were a common pattern (χ 2, P 0.05). Absolute values of albumin, α1, α2, β, and γ globulin were 2.9 ± 0.24, 0.97 ± 0.19, 0.96 ± 0.24, 1.2 ± 0.36, and 1.3 ± 0.29 g/dl, respectively. Mean albumin to globulin ratio was 0.67. In conclusion, analysis of serum proteins in the miniature Caspian horse using CAE showed five protein fractions including albumin, α1, α2, β, and γ globulin.

Intestinal impaction by Parascaris equorum in Caspian miniature horse in Kerman, Iran

Caspian miniature horses are extremely rare and have barely survived. The large roundworm of horses, Parascaris equorum, is an important cosmopolitan nematode parasite of foals. Two Caspian miniature horses (3 and 5 years old) from one herd with clinical sings of colic were referred to hospital of Veterinary Medicine Faculty of Kerman University, from July 2008 to March 2009. Fecal samples were taken from each horse and examined with the Cornell-McMaster dilution technique. Parascaris eggs were observed, counted, and the eggs per gram feces were calculated. At necropsy, the carcasses were cachexic, and there were massive burden of P. equorum in the small intestine.

Growth characteristics of fibroblasts isolated from the body and limb of the Caspian miniature horse and the effect of hydrocortisone in vitro

The present study was designed to determine the in vitro effects of hydrocortisone on the growth of fibroblasts isolated from both the neck and distal limb of the Caspian miniature horse. Three grammes of subcutaneous tissue was harvested from the neck and metacarpi of four Caspian miniature horses and tissue from each site was placed in a separate culture medium. After separation of fibroblasts using explant culture method, a concentration of 10,000 cells/ml was placed into each well of a 24-well plate. Hydrocortisone was added in three different concentrations (control group: without hydrocortisone, treatment groups A, 10; B, 100 and C, 300 μg/ml) into the culture medium containing fibroblasts, 4 days later the cells were counted. The effects of hydrocortisone on fibroblasts growth and viability were evaluated. Results show that the mean fibroblast growth rate from neck-derived fibroblasts was significantly greater than those derived from the limb. There were no significant differences in fibroblast growth rate between the control group and group A in both neck and limb-derived fibroblast (P > 0.05), but groups B and C suppressed fibroblast growth significantly in comparison with their related control groups (P < 0.05). Cell viability decreased significantly in treatment groups B and C in comparison with the control group in both neck and limb-derived fibroblasts. The mean percentage of fibroblast growth inhibition from the neck and limb significantly decreased in treatment groups B and C in comparison with their related control groups. The effect of hydrocortisone on cultured fibroblast growth was dose-dependant.

Analysis of genetic diversity and estimation of inbreeding coefficient within Caspian horse population using microsatellite markers.

The present study was undertaken to genetically evaluate Caspian horses for genetic diversity and to asses whether they have experienced recent population bottlenecks. A total of 100 individuals were characterized for within breed diversity using 16 microsatellite markers. The estimated mean number of alleles was 8.69 per locus, with a total of 139 alleles in the genotyped sample. The mean effective number of alleles in the Caspian horse population was 5.86, ranging from 3.49 to 8.49. The average observed heterozygosity in the present study (0.52) was lower than to the expected heterozygosity (0.82), which may reflect the narrow genetic base of the current population of this breed. All marker loci employed in this study were very informative with an average of 0.80. The Chi-square and likelihood ratio tests performed to examine population for HWE showed some highly significant deviations from HWE. Estimated values of Wright’s fixation index, FIS (0.367) indicates a certain level of heterozygote deficiency. A significant heterozygote excess on the basis of different models, as revealed from Sign and Wilcoxon rank test suggested that Caspian horse population is not in mutation-drift equilibrium. But, the Mode-shift indicator test showed a normal ‘L’ shaped distribution for allelic class and proportion of alleles, thus indicating the absence of bottleneck events in the recent history of this breed. The present work is a contribution to the knowledge of population structure and to the assessment of genetic diversity that may be helpful to horse breeders in designing and managing breeding or conservation strategies for the Caspian horse breed.

Bottleneck Study and Genetic Structure of Iranian Caspian Horse Population Using Microsatellites

Genetic diversity within the Iranian Caspian horse was evaluated using 8 different microsatellite pairs on 45 Caspian horse blood samples. This molecular characterisation was undertaken to evaluate the problem of genetic bottlenecks, if any, in this breed. The number of alleles per locus varied from 3 to 5 with mean value of 4.125. All markers have relatively high PIC value (> 0.6), observed heterozygosity; 0.9433, expected Levene's heterozygosity 0.6856 and expected Nei's heterozygosity equal to 0.6762. This study indicated the existence of substantial genetic diversity in the Caspian horse. No significant genotypic linkage disequilibrium was detected across the population, suggesting no evidence of linkage between loci. A mode-shifted distribution, significant heterozygote excess on the basis of different mutation models, as revealed from Sign, Standardized differences and Wilcoxon rank tests suggested that there was recent bottleneck in the existing Caspian horse. Urgent conservational strategies on this population, is recommended.