Chinese Donkey Research Articles

First identification and isolation of equine herpesvirus type 1 in aborted fetal lung tissues of donkeys.

Equine herpesvirus type 1 (EHV-1) is commonly associated with horse abortion. Currently, there are no reported cases of abortion resulting from EHV-1 infection in donkeys. This was the first survey-based study of Chinese donkeys. The presence of EHV-1 was identified by PCR. This survey was conducted in Chabuchar County, North Xinjiang, China, in 2020. A donkey EHV-1 strain (Chabuchar/2020) was successfully isolated in MDBK cells. Seventy-two of 100 donkey sera were able to neutralize the isolated EHV-1. Moreover, the ORF33 sequence of the donkey-origin EHV-1 Chabuchar/2020 strain showed high levels of similarity in both its nucleotide (99.7‒100%) and amino acid (99.5‒100%) sequences, with those of horse EHV-1 strains. EHV-1 Chabuchar/2020 showed significant consistency and was classified within cluster 1 of horse EHV-1 strains. Further, analysis of the expected ORF30 nucleotide sequence revealed that donkey EHV-1 strains contained guanine at position 2254, resulting in a change to aspartic acid at position 752 of the viral DNA polymerase. Therefore, these strains were classified as horse neuropathogenic strains. Lastly, a phylogenetic tree was constructed using the partial ORF68 nucleotide sequences, showing that the identified donkey EHV-1 strain and the EHV-1 strain found in aborted Yili horses in China comprised a novel independent VIII group. This study showed the first isolation and identification of EHV-1 as an etiological agent of abortions in donkeys. Further analysis of the ORF33, ORF30, and ORF68 sequences indicated that the donkey EHV-1 contained the neuropathogenic genotype of strains in the VIII group. It is thus important to be aware of EHV-1 infection in the donkey population, even though the virus has only been identified in donkey abortions in China.

Analysis of gut microbiota in chinese donkey in different regions using metagenomic sequencing

BackgroundGut microbiota plays a significant role in host survival, health, and diseases; however, compared to other livestock, research on the gut microbiome of donkeys is limited.ResultsIn this study, a total of 30 donkey samples of rectal contents from six regions, including Shigatse, Changdu, Yunnan, Xinjiang, Qinghai, and Dezhou, were collected for metagenomic sequencing. The results of the species annotation revealed that the dominant phyla were Firmicutes and Bacteroidetes, and the dominant genera were Bacteroides, unclassified_o_Clostridiales (short for Clostridiales) and unclassified_f_Lachnospiraceae (short for Lachnospiraceae). The dominant phyla, genera and key discriminators were Bacteroidetes, Clostridiales and Bacteroidetes in Tibet donkeys (Shigatse); Firmicutes, Clostridiales and Clostridiales in Tibet donkeys (Changdu); Firmicutes, Fibrobacter and Tenericutes in Qinghai donkeys; Firmicutes, Clostridiales and Negativicutes in Yunnan donkeys; Firmicutes, Fibrobacter and Fibrobacteres in Xinjiang donkeys; Firmicutes, Clostridiales and Firmicutes in Dezhou donkeys. In the functional annotation, it was mainly enriched in the glycolysis and gluconeogenesis of carbohydrate metabolism, and the abundance was the highest in Dezhou donkeys. These results combined with altitude correlation analysis demonstrated that donkeys in the Dezhou region exhibited strong glucose-conversion ability, those in the Shigatse region exhibited strong glucose metabolism and utilization ability, those in the Changdu region exhibited a strong microbial metabolic function, and those in the Xinjiang region exhibited the strongest ability to decompose cellulose and hemicellulose.ConclusionAccording to published literature, this is the first study to construct a dataset with multi-regional donkey breeds. Our study revealed the differences in the composition and function of gut microbes in donkeys from different geographic regions and environmental settings and is valuable for donkey gut microbiome research.

Effect of two extenders on the cryopreservation of Biyang donkey semen

The Biyang donkey is a medium-sized Chinese donkey. Very little research has been done on semen cryopreservation in this donkey breed. Dimethylformamide (DMF) and ethylene glycol (EG) are small molecular weight osmotic cryoprotectants that enter equine spermatozoa more readily and effectively than glycerol, and spermatozoa have reduced swelling mortality when equilibrated in amide-containing extenders. In recent years, EG and DMF have been used in extenders to improve the quality of cryopreserved donkey semen. It is, however, unknown whether their combination with glycerol is more effective than glycerol alone in protecting the semen of Biyang donkeys. In this experiment, we aimed to compare the effects of two different semen extenders on semen cryopreservation in Biyang donkeys. Both extenders were based on the CAU1 extender (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). The cryoprotectant 1 consisted of 2.5% egg yolk and 2.5% glycerol in CAU1 extender, cryoprotectant 2 consisted of 2.5% yolk, 1.5% DMF, 0.5% glycerin and 0.5% EG in CAU1 extender. In five donkeys, semen was collected three times to deplete epididymalsperm reserves, then one ejaculation of each jackass was collected for semen freezing was described (Feng Yang et.al. Equine Veterinary Journal 2021, 53: 1218-1226). Briefly, after the semen was collected, an equal volume of CAU1 dilution was added and left to stand at room temperature (22-25°C) for 30 min, thereafter semen was divided into two equal portions and centrifuged to remove the semen plasma, one was diluted with cryoprotectant 1 and the other with cryoprotectant 2. Diluted semen was filled into 0.5 mL straws with a filling machine and left to stand at 4°C for 2-3 hours. Cryopreservation was carried out at a height of 3-5 cm above liquid nitrogen for 15 min, straws were then stored in liquid nitrogen. Two days later, semen was thawed in a water bath at 37°C for 40 seconds. Sperm motility was examined by CASA (TYCASA1.0, Beijing Tianyuan Aorui Biotechnology Co., Ltd). Motility of the raw semen (n=5) was 89.6±4.0%. The frozen-thawed motility was 56.0±4.18% and 51.6±4.3% in cryoprotectant 1 and cryoprotectant 2, respectively (not significant). In conclusion, the two cryo-extenders were equally suitable for cryopreservation of Biyang donkey semen.

A survey report on the donkey original breeding farms in China: Current aspects and future prospective.

The number of the large-scale donkey breeding farms in China has increased dramatically. However, information regarding the situation of a Chinese donkey population under large-scale donkey breeding farms is limited. This survey report was conducted using questionnaires online to investigate the current situation of the donkey original breeding farms in China, in terms of donkey stock, local breeds, reproductive parameters, growth and lactation performance, and future perspectives. China has developed the donkey reserve system based on national, provincial and non-governmental (self-own) donkey original breeding farms. In the present study, a total of 38 donkey original breeding farms concentrated in Northern of China were studied, and 52% of them keep their donkeys with a stocking density of 100-500 donkeys. China is rich in various local donkey breeds, and 16 local donkey breeds including large-sized, medium-sized and small-sized breeds were collected in our survey. Dezhou donkey are prevalent with a percentage of more than 57% of the total donkeys, while the Cullen donkeys belong to small-sized donkey breeds are scare. The reproductive efficiency and donkey productivity were different across donkey farms, indicating potential differences in management and breeding practices between different donkey original breeding farms. The artificial insemination has been performed in these donkey farms with an average proportion of 73%. Regarding the donkey productivity, the national and provincial donkey original breeding farms showed a higher birthweight and fat content in donkey milk than self-own farms. Furthermore, our results indicate that the donkey breeds with different body size also have important influence on the reproduction parameters and donkey productivity, with the large-sized donkeys had better performance compared to the small-sized donkeys. In summary, our survey provided valuable baseline information on the situation of donkey population dynamics in the donkey original breeding farms. However, further study is required in the future to investigate the factors such as donkey health care, management and nutrition during breeding, fattening and lactation that influence donkey productivity under large-scale farm systems.

Morphological and molecular characterization of a Sarcocystis species infecting donkeys from China.

There is considerable confusion concerning the relationships among species of Sarcocystis found in donkeys and horses. Here, we describe a Sarcocystis species in Chinese donkeys (Equus asinus). Sarcocysts were found in 12 of 32 (37.5%) adult donkeys. By light microscopy, they were divided into two types, thin-walled and thick-walled. The thin-walled were macroscopic (up to 320μm wide) and had short club-like protrusions (up to 2.7μm long); the thick-walled were microscopic (up to 135μm wide) and had villar protrusions (up to 5.4μm long). Ultrastructures of the two types exhibited similar morphological characteristics, including bundled microtubules in the core of the villar protrusions penetrating diagonally into the ground substance, similar to wall type 11c. Three genetic markers, 18S rDNA, 28S rDNA, and mitochondrial cox1, obtained from the two morphotypes were sequenced and analyzed. The sequences of the three loci in the two morphotypes presented high intraspecific similarities of 97.2-99.5%, 97.8-99.6% and 99.0 - 99.9%, respectively. The most similar sequences in GenBank to the newly obtained 18S rDNA, 28S rDNA and cox1 sequences were those of Sarcocystis spp. in horses, with similarities of 90.0 - 97.5%, 94.7 - 95.1%, and 82.6 - 84.5%, respectively. Phylogenetic analysis using the three genetic markers indicated that the Sarcocystis sp. in donkeys formed an individual clade most closely related to a clade encompassing Sarcocystis spp. in horses. Further studies are needed for taxonomic identification of sarcocysts in donkeys because the Sarcocystis species in donkeys and horses are not successfully cross transmissible despite morphological similarities.

Genetic architectures and selection signatures of body height in Chinese indigenous donkeys revealed by next-generation sequencing.

Donkeys are widely distributed labour animals in the world. During the process of the domestication and artificial selection of domestic donkeys, body sizes show significant differences among different breeds of donkeys. Based on the genome resequencing data of 103 Chinese indigenous donkeys from 11 breeds (Biyang, Dezhou, Guangling, Hetian, Jiami, Kulun, Qingyang, Turfan, Tibetan, Xinjiang, and Yunnan), seven Spanish donkeys from two breeds (Zamorano~Leonés and Andalusian), and three wild donkeys, we investigated the population structures of Chinese domestic donkeys with different body sizes. We used FST and XP-EHH analyses to explore the selected regions related to body sizes. The results showed that Chinese indigenous donkeys have a closer relationship with African wild donkeys than with Asian wild donkeys. LCORL/NCAPG, FAM184B, TBX3, and IHH were identified as genes with strong signals in analysis of selection signature (FST and XP-EHH) in large and small donkeys. The seven identified variants can be served as candidate loci affecting the body size of Chinese donkeys. Five of seven loci were located in intron 9 of FAM184B and were in a haplotype block, and one of the identified variants (Chr03:112664848) located in the CDS region of the LCORL gene was found to cause stop-loss. These candidate genes and variants shed new light on the molecular basis of donkey body size and will facilitate the breeding activities of donkeys.

Single nucleotide polymorphisms (SNPs) and indels identified from whole-genome re-sequencing of four Chinese donkey breeds

This paper represents the fundamental report of the survey of genome-wide changes of four Chinese indigenous donkey breeds, Dezhou (DZ), Guangling (GL), North China (NC), and Shandong Little donkey (SDL), and the findings will prove usefully for identification of biomarkers that perhaps predict or characterize the growth and coat color patterns. Three genomic regions in CYP3A12, TUBGCP5, and GSTA1 genes, were identified as putative selective sweeps in all researched donkey populations. The loci of candidate genes that may have contributed to the phenotypes in body size (ACSL4, MSI2, ADRA1B, and CDKL5) and coat color patterns (KITLG and TBX3) in donkey populations would be found in underlying strong selection signatures when compared between large and small donkey types, and between different coat colors. The results of the phylogenetic analysis, F ST, and principal component analysis (PCA) supported that each population cannot clearly deviate from each other, showing no obvious population structure. We can conclude from the population history that the formation processes between DZS and NC, GL, and SDL are completely different. The genetic variants discovered here provide a rich resource to help identify potential genomic markers and their associated molecular mechanisms that impact economically important traits for Chinese donkey breeding programs.

Genomic analyses reveal distinct genetic architectures and selective pressures in Chinese donkeys

Donkey (Equus asinus) is an important livestock animal in China because of its draft and medicinal value. After a long period of natural and artificial selection, the variety and phenotype of donkeys have become abundant. We clarified the genetic and demographic characteristics of Chinese domestic donkeys and the selection pressures by analyzing 78 whole genomes from 12 breeds. According to population structure, most Chinese domestic donkeys showed a dominant ancestral type. However, the Chinese donkeys still represented a significant geographical distribution trend. In the selective sweep, gene annotation, functional enrichment, and differential expression analyses between large and small donkey groups, we identified selective signals, including NCAPG and LCORL, which are related to rapid growth and large body size. Our findings elucidate the evolutionary history and formation of different donkey breeds and provide theoretical insights into the genetic mechanism underlying breed characteristics and molecular breeding programs of donkey clades.

Brazilian donkey slaughter and exports from 2002 to 2019

The international demand for donkeys has increased sharply in recent years, motivated by China’s growing interest in producing ejiao (a traditional medicine made from donkey skin) and, in a smaller proportion, donkey meat. Since the Chinese donkey population dropped by 75.4% in the past 25 years, the country has searched for supply in the international market, mainly in Africa and South America. Aiming to understand the participation of Brazil in this scenario, this paper presents and discusses official data on donkey slaughter and donkey meat and skin exports in Brazil from 2002 to 2019. After the recent news reports of donkey trade-related mistreatment in the Brazilian Northeast, the issue has attracted attention from public authorities, the media, and civil society, but it lacked a quantitative description that could give a tangible dimension to the situation. The interpretation of these data is made with the collaboration of qualitative research methods, emphasizing some aspects of human-animal relations.

A Survey of Smallholder Farms Regarding Demographics, Health Care, and Management Factors of Donkeys in Northeastern China.

Essential information on the population dynamics and the health and welfare of Chinese donkeys is scarce. The objectives of this study were to describe the demographic characteristics, management and health care of a sample of donkeys under smallholder farm conditions of northeastern China. A cross-sectional survey of 731 randomly selected donkey owners on smallholder farms (1,658 donkeys) in 40 villages of northeastern China was conducted. Data on the composition and management of the donkeys and their routine health care were analyzed. The surveyed donkey population consisted of mostly (83.8%) jenny/filly donkeys with a mean age of 6.2 ± 5.0 years. Most (91.2%) of the farms kept 1–4 donkeys. The majority of donkeys were used for breeding and labor. Most (93.8%) of the farms did not have bedding, and their mean stable size was 17.7 ± 10.1 m2. All of the animals were turned out for at least part of the year. The mean size of the turnout areas on the farms was 17.8 m2. The condition of 12.5% of the donkeys was evaluated as “poor” with a body condition score of 1 on a scale of 5. More than one third (37.9%) of the donkeys had never been dewormed. Also, none of them were ever vaccinated or received dental care from a veterinarian. Their hoofs were trimmed once (45.9%) or twice (27.6%) a year. Forty percent of the donkeys were reported to suffer from at least one medical problem in the preceding year. The most common medical problems were colic, respiratory disorders and skin conditions. Owners seemed to underestimate some of the most prevalent diseases in donkeys, suggesting that their knowledge of the management of donkeys, including routine healthcare practices should be improved to ensure the health and welfare of donkeys in northeastern China.

Detection of Selection Signatures Underlying Production and Adaptive Traits Based on Whole-Genome Sequencing of Six Donkey Populations.

Simple SummaryAfter a long period of artificial selection, the donkey now presents a variety of body types and production performance values. In this experiment, we performed selective signal scanning on the second-generation resequencing data of six different varieties. The regions and candidate genes related to artificial selection were identified to provide reference for future breeding.Donkeys (Equus asinus) are an important farm animal. After long-term natural and artificial selection, donkeys now exhibit a variety of body sizes and production performance values. In this study, six donkey breeds, representing different regions and phenotypes, were used for second-generation resequencing. The sequencing results revealed more than seven million single nucleotide variants (SNVs), with an average of more than four million SNVs per species. We combined two methods, Z-transformed heterozygosity (ZHp) and unbiased estimates of pairwise fixation index (di) values, to analyze the signatures of selection. We mapped 11 selected regions and identified genes associated with coat color, body size, motion capacity, and high-altitude adaptation. These candidate genes included staining (ASIP and KITLG), body type (ACSL4, BCOR, CDKL5, LCOR, NCAPG, and TBX3), exercise (GABPA), and adaptation to low-oxygen environments (GLDC and HBB). We also analyzed the SNVs of the breed-specific genes for their potential functions and found that there are three varieties in the conserved regions with breed-specific mutation sites. Our results provide data to support the establishment of the donkey SNV chip and reference information for the utilization of the genetic resources of Chinese domestic donkeys.

Genetic diversity and relationships of Chinese donkeys using microsatellite markers.

Donkeys are one important livestock in China because of their nourishment and medical values. To investigate the genetic diversity and phylogenetic relationships of Chinese donkey breeds, a panel of 25 fluorescently labeled microsatellite markers was applied to genotype 504 animals from 12 Chinese donkey breeds. A total of 226 alleles were detected, and the expected heterozygosity ranged from 0.6315 (Guanzhong) to 0.6999 (Jiami). The mean value of the polymorphism information content, observed number of alleles, and expected number of alleles for all the tested Chinese donkeys were 0.6600, 6.890, and 3.700, respectively, suggesting that Chinese indigenous donkeys have relatively abundant genetic diversity. Although there were abundant genetic variations found, the genetic differentiation between the Chinese donkey breeds was relatively low, which displayed only 5.99 % of the total genetic variance among different breeds. The principal coordinates analysis clearly splits 12 donkey breeds into two major groups. The first group included Xiji, Xinjiang, Liangzhou, Kulun, and Guanzhong donkey breeds. In the other group, Gunsha, Dezhou, Biyang, Taihang, Jiami, Qingyang, and Qinghai donkeys were clustered together. This grouping pattern was further supported by structure analysis and neighbor-joining tree analysis. Furthermore, genetic relationships between different donkey breeds identified in this study were corresponded to their geographic distribution and breeding history. Our results provide comprehensive and precise baseline information for further research on preservation and utilization of Chinese domestic donkeys.

Characterization of the complete mitochondrial genome of the Liangzhou donkey (Equus asinus)

Liangzhou donkey (Equus asinus) is one of the most important donkey species in China. In this study, we sequenced the complete mitochondrial genome by next-generation sequencing method, the results show it was 16,560 bp in length, consisting 37 genes: 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a non-coding control region. The base composition of the genome was 32.4% for A, 28.7% for C, 13.2% for G, and 25.7% for T； A + T for 58.1%. The phylogenetic analysis demonstrated that Liangzhou donkey was more closely related to Qingyang donkey and processed distinct relationships with other Chinese donkey breeds. This work characterized the mitochondrial genome of Equus asinus (Liangzhou donkey) and presents significant genetic information for this special species.

Copy number variations of five Y chromosome genes in donkeys

Abstract. In mammals, the Y chromosome plays a pivotal role in male sex determination and is essential for normal sperm production. A number of studies were conducted on Y chromosome genes of various species and identified single-copy and multi-copy genes. However, limited studies about donkey Y chromosome genes have been done. In this study, 263 male samples from 13 Chinese donkey breeds were collected to analyze the copy number variations (CNVs) of five Y chromosome genes using the quantitative PCR (qPCR) method. These five genes (cullin 4 B Y (CUL4BY), equus testis-specific transcript y1 (ETSTY1), equus testis-specific transcript y4 (ETSTY4), equus testis-specific transcript Y 5 (ETSTY5), and sex-determining region Y (SRY) were identified as multi-copy, whose median copy numbers (MCNs) were 5, 45, 2, and 2, and 13 with CNV ranges of 1–57, 1–227, 1–37, 1–86 and 1–152, respectively. The CNVs of these five genes were shared in different breeds. Compared to previous studies, the copy numbers of five genes showed some distinct consequences in this study. In particular, the well-known single-copy SRY gene showed CNVs in donkeys. Our results provided genetic variations of donkey Y chromosome genes.

Evaluation of genetic diversity and population structure of five Chinese indigenous donkey breeds using microsatellite markers

China had the largest population of raising donkeys in the world, however the number of Chinese indigenous donkey decreased dramatically due to the increase of agriculture mechanization in the last century. The species has still been important in China because of its edible and medical value, therefore the survey on its genetic diversity in China is necessary for its conservation and utilization. In this study, 15 microsatellite markers were used to evaluate genetic diversity and population structure of five Chinese indigenous donkey breeds. The mean values of expected heterozygosity, allelic richness, and total number of alleles for all the tested Chinese donkeys were 0.70, 6.04, and 6.28 respectively, suggesting that the genetic diversity of Chinese indigenous donkeys is rich. The Bayesian analysis and principal component analysis plot yielded the same clustering result, which revealed that Guanzhong donkey was the most differentiated breed in all detected samples, and Jinnan (JN) and Guangling (GL) were genetically closed together. Additionally, our results indicated that the heterozygote deficit was severe in two Chinese indigenous donkey breeds (GL and JN), and it warned us that animal conservation activities on this species should be considered carefully in near future.

The genetic diversity analysis in the donkey myostatin gene

Myostatin (MSTN) gene negatively controls skeletal muscle development and growth, variations of which play an important role in the regulation of skeletal muscle growth in mammals. However, study on genetic polymorphism ofMSTN gene in donkey is limited. In this study, we screened the single nucleotide polymorphsims (SNPs) ofMSTN gene in 13 Chinese donkey breeds. Four novel SNPs (g.229T>C, g.872A>G, g.2014G>A, and g.2395C>G) were detected and genotyped by sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Six haplotypes (H1-H6) were analyzed, which indicated abundant haplotype diversities in Chinese donkeys. The haplotype H1 was the most dominant and ancient in all breeds. Xinjiang donkey displayed the highest haplotype diversity. The Neighbour-Joining (NJ) tree ofMSTN gene among different species was constructed. The clustering result of nine species was consistent with the fact of species differentiation. Our results will provide a reliable theoretical basis for the preservation, exploration and utilization of Chinese donkey genetic resources.

ASIP gene variation in Chinese donkeys.

ASIP gene variation in Chinese donkeys.

Complete mitochondrial genome of Qingyang donkey (Equus asinus)

The present study aimed to report the entire mitochondrial genome of Qingyang donkey (Equus asinus). Donkeys play an important role in the daily lives of human beings. The population of Chinese donkey breeds has undergone an enormous decline with the advance of agricultural mechanization, rapid development of transportation and introduction of new different breeds over the past few decades. As a result, a large number of donkey breeds are close to extinction, especially Qingyang donkey. Mitochondrial DNA is used as a molecular tool in research on biodiversity conservation and sustainable utilization of biological resources. The circular genome sequenced using the Illumina Hiseq 2500 platform was 16,568 base pairs (bp), containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes and a non-coding control region. The initiation site of the L-strand replication origin, 32 bp in length, was between tRNA-Asn and tRNA-Cys. The base composition of the genome was A (32.4%), C (28.7%), G (13.2%) and T (25.7%) with an A+T content of 58.1%. Molecular phylogeny demonstrated two separated clades in Equidae and the presence of a distant relationship between the domestic donkey breeds and Asian wild asses, indicating that domestic donkeys might not probably have originated from Asian wild asses. However, African wild asses were grouped as a sister to domestic donkeys. This study presented the complete mitochondrial genome of E. asinus, providing the important genetic background of protection and adequate utilization of domestic donkey breeds.

High polymorphism at microsatellite loci in the Chinese donkey.

To reveal the genetic diversity and phylogenetic relationships between Chinese donkey breeds, 415 individuals representing ten breeds were investigated using ten microsatellite markers. The observed number of alleles, mean effective number of alleles (NE), mean expected heterozygosity (HE), and polymorphic information content (PIC) of each breed and polymorphic locus were analyzed. The results showed that seven (HTG7, HTG10, AHT4, HTG6, HMS6, HMS3, and HMS7) of ten microsatellite loci were polymorphic. The mean PIC, HE, and NE of seven polymorphic loci for the ten donkey breeds were 0.7679, 0.8072, and 6.0275, respectively. These results suggest that domestic Chinese donkey breeds possess higher levels of genetic diversity and heterozygosity than foreign donkeys. A neighbor-joining tree based on Nei's standard genetic distance showed that there was close genetic distance among Xinjiang, Qingyang, Xiji, and Guanzhong donkey breeds. In addition, Mongolia and Dezhou donkey breeds were placed in the same category. The phylogenetic tree revealed that the genetic relationships between Chinese donkey breeds are consistent with their geographic distribution and breeding history.

Ancient DNA provides new insight into the maternal lineages and domestication of Chinese donkeys.

BackgroundThe donkey (Equus asinus) is an important domestic animal that provides a reliable source of protein and method of transportation for many human populations. However, the process of domestication and the dispersal routes of the Chinese donkey are still unclear, as donkey remains are sparse in the archaeological record and often confused with horse remains. To explore the maternal origins and dispersal route of Chinese donkeys, both mitochondrial DNA D-loop and cytochrome b gene fragments of 21 suspected donkey remains from four archaeological sites in China were amplified and sequenced.ResultsMolecular methods of species identification show that 17 specimens were donkeys and three samples had the maternal genetic signature of horses. One sample that dates to about 20,000 years before present failed to amplify. In this study, the phylogenetic analysis reveals that ancient Chinese donkeys have high mitochondrial DNA diversity and two distinct mitochondrial maternal lineages, known as the Somali and Nubian lineages. These results indicate that the maternal origin of Chinese domestic donkeys was probably related to the African wild ass, which includes the Nubian wild ass (Equus africanus africanus) and the Somali wild ass (Equus africanus somaliensis). Combined with historical records, the results of this study implied that domestic donkeys spread into west and north China before the emergence of the Han dynasty. The number of Chinese domestic donkeys had increased primarily to meet demand for the expansion of trade, and they were likely used as commodities or for shipping goods along the Silk Road during the Tang Dynasty, when the Silk Road reached its golden age.ConclusionsThis study is the first to provide valuable ancient animal DNA evidence for early trade between African and Asian populations. The ancient DNA analysis of Chinese donkeys also sheds light on the dynamic process of the maternal origin, domestication, and dispersal route of ancient Chinese donkeys.