The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Intrageneric amplification of horse microsatellite markers with emphasis on the Przewalski's horse (E. pmewalskii)

Primer sequences flanking 13 microsatellite loci isolated from the domestic horse (E. caballus) were successfully used to amplify homologous loci in the Przewalski's horse (E. przewalskii). The results demonstrate that the level of polymorphism at all 13 loci in the Przewalski's horse was comparable to that in the domestic horse and the overall exclusion probability in the Przewalski's horse was calculated to be 0.9994. The results suggest that it should be possible to use E. caballus-derived microsatellite markers to provide parentage verification and additional valuable information to the captive management of E. przewalskii. The ability to amplify corresponding loci in the remaining five species of the genus was also confirmed, illustrating the general application of markers isolated from the domestic horse to the evaluation of polymorphism in the other six species of the genus.     

The application of artificial reproduction techniques to the propagation of selected endangered species

Artificial reproduction techniques were applied to individual animals not capable of natural reproduction. All individuals represented endangered species. An aged and debilitated Arabian oryx was treated with prostaglandin F2 α (PG) and pregnant mare serum gonadotropin (PMSG) prior to euthanasia. She responded to the treatment with mild superovulation. Four follicular oocytes were recovered, all of which subsequently matured in vitro and were frozen. A group of three Soemmering's gazelles, including a crippled male and a three-legged female, was evaluated for reproductive potential. The male was electro-ejaculated and determined to be producing normal sperm; semen was frozen. Estrus was induced and synchronized in the two females with PMSG and PG prior to the introduction of the male. Copulation attempts were unsuccessful. A second modified estrus induction attempt did not result in behavioral estrus in either female. The handicapped female was subsequently superovulated with PMSG before euthanasia. Four oocytes were recovered but failed to mature in vitro. Two Przewalski's horse mares are not able to mate naturally due to chronic laminitis. Preliminary studies have been undertaken to determine, through ultrasound examination, the estrous cycles and ovarian dynamics of the two horses, in preparation for artificial insemination and embryo transfer. Cycle length and variability, ovarian response to PG and rate of follicle growth have been determined to be similar to the domestic horse.     

Improvement of attachment rate of bovine embryos after transfer with trophoblastic fragments

Fragments of trophoblastic vesicles have been shown to improve embryo development when used in co-culture. We wished to investigate the effect of trophoblastic fragments on establishment of pregnancy after embryo transfer. Embryos (n=129) frozen by direct immersion in liquid nitrogen (Group 1), 82 embryos frozen by the slow freezing method (Group 2) and 55 fresh embryos stored at 20-22 degrees C for 21-22 h (Group 3) were transferred into recipient heifers. The embryos of each group were divided in 2 batches. Batch 1 embryos (control) were transferred without trophoblastic fragments, Batch 2 embryos of each group (experimental) were transferred in combination with frozen-thawed trophoblastic fragments at a rate of 5 to 7 fragments per embryo. The fragments were produced from 16-17 day bovine embryos frozen by vitrification. Two months following the embryo transfers it was established that the attachament rate for Batch 1 embryos in Groups 1, 2, 3 was 33%, 39% and 48%, respectively; for Batch 2 embryos in the same Groups the rate was 55%, 56% and 74%, respectively. We conclude that trophoblastic fragments promote embryo attachment, but their effect does not continue beyond the time of attachment.     

Switched identity of two Przewalski's horse mares detected by blood typing

Accurate pedigrees are of prime importance to breeders of both domestic and captive animals. Blood typing is a tool utilized to verify accuracy of pedigrees in domestic animal breeding programs that may often be directly applied to related zoo species. We describe the application of blood typing techniques developed for domestic horses to detect a switch in the identity of two Przewalski's horse mares. Before the switch detection, one of the mares had been bred to a closely related stallion, a breeding which would not have been planned had the correct identity of the mare been known.     

放归普氏野马核心区黑腹胃蝇疫源分布特点

黑腹胃蝇是新疆荒漠草原优势种马胃蝇,对放归至当地普氏野马构成了严重威胁。该蝇将卵产在牧草上并以此感染采食者——马科动物,其幼虫于宿主消化道营寄生生活,至三龄老熟幼虫随宿主粪便进入环境化蛹,成为当年新的疫源。于黑腹胃蝇幼虫两个发生峰期(4—5月和8月)对普氏野马核心区新鲜粪便和黑腹胃蝇幼虫点位进行了调查,采用最小凸多边形法(MCP)和固定核域法(FKE)计算粪便分布范围,并通过核密度分析(KDE)辨识幼虫聚集区域和特征。结果表明：(1)在黑腹胃蝇种群第一、第二峰期,野马采食草场拥有最多的胃蝇幼虫采集占比(57.25%、41.94%),其次是驴道、山坡、水源地区域;(2)最小凸多边形法(95%利用率)和固定核域法(95%利用率、0.006带宽值)均能较好估算野马粪便分布范围,第一峰期普氏野马粪便范围(60—70 km²)稍大于第二峰期(50—60 km²)。在所调查的八个水源地,两个发生高峰期均包含红柳、5号、6号3个水源地,并形成了以水源地为重要支撑点、依托毗邻草场交叉传播的连续、非均匀疫源地分布特点;(3)水源地附近草场具有更高的黑腹胃蝇幼虫...     

Asian horses deepen the MSY phylogeny

Humans have shaped the population history of the horse ever since domestication about 5500 years ago. Comparative analyses of the Y chromosome can illuminate the paternal origin of modern horse breeds. This may also reveal different breeding strategies that led to the formation of extant breeds. Recently, a horse Y‐chromosomal phylogeny of modern horses based on 1.46 Mb of the male‐specific Y (MSY) was generated. We extended this dataset with 52 samples from five European, two American and seven Asian breeds. As in the previous study, almost all modern European horses fall into a crown group, connected via a few autochthonous Northern European lineages to the outgroup, the Przewalski's Horse. In total, we now distinguish 42 MSY haplotypes determined by 158 variants within domestic horses. Asian horses show much higher diversity than previously found in European breeds. The Asian breeds also introduce a deep split to the phylogeny, preliminarily dated to 5527 ± 872 years. We conclude that the deep splitting Asian Y haplotypes are remnants of a far more diverse ancient horse population, whose haplotypes were lost in other lineages.     

Ancient Genomes Reveal Unexpected Horse Domestication and Management Dynamics

The horse was essential to past human societies but became a recreational animal during the twentieth century as the world became increasingly mechanized. As the author reviews here, recent studies of ancient genomes have revisited the understanding of horse domestication, from the very early stages to the most modern developments. They have uncovered several extinct lineages roaming the far ends of Eurasia some 4000 years ago. They have shown that the domestic horse has been significantly reshaped during the last millennium and experienced a sharp decline in genetic diversity within the last two centuries. At a time when no truly wild horses exist any longer, this calls for enhanced conservation in all endangered populations. These include the Przewalski's horse native to Mongolia, and the many local breeds side-lined by the modern agenda, but yet representing the living heritage of over five millennia of horse breeding.     

野放普氏野马(Equus przewalskii)家域面积及其影响因素

采用MCP方法研究了2011年至2012年新疆卡拉麦里山有蹄类自然保护区野放普氏野马家域的变化。通过方差分析验证了年间、季节间不同群体家域及其两两重叠无差异。以家族群大小为协变量进行了野放野马家域协方差分析。利用野放野马家族大小为协变量的协方差分析分析检验了野放野马家族大小与家域关系。结果表明:(1)野马平均家域面积由2011年的(20±2)km~2/匹扩大到2012年的(30±2)km~2/匹。对部分野放群体中头马未发生更替的野马群的研究表明,随着野马群体增大,其家域面积显著增大(P0.05)。(2)单因素方差分析显示,不同野马群的家域面积在不同年份差异显著,且春季家域秋季家域夏季家域。(3)2011年不同群家域两两间相互重叠面积与群大小无显著相关(r=0.256,P=0.5800.05)。而2012年野马群家域两两之间重叠面积有显著差异(F=4.521,df=8,P0.001)。家域两两相互重叠面积与群大小显著相关(r=0.706,P=0.0330.05)。(4)不同季节间野马群家域重叠面积有显著差异(F=5.695,df=8,P0.001)。5号群、7号群和8号群的自身家域重叠面积(P0.05),3号群、6号群和9号群的家域重叠面积(P0.05)。(5)影响野放野马家域面积的生物因子有草本盖度、灌木盖度,非生物因子主要有温度、湿度、风速、最近水源地距离和最近居民点距离等。温度与草本盖度是影响野放野马家域面积大小的主要因素,两者与野放野马家域面积显著相关(P0.01)。     

Successful transfer of the embryos of Przewalski's horses (Equus przewalskii) and Grant's zebra (E. burchelli) to domestic mares (E. caballus)

Blastocysts were collected non-surgically from 2 Przewalski's horse and 2 Grant's zebra mares and transferred extra-specifically to domestic horse and donkey recipients. Nine Przewalski's horse embryos were transferred surgically, and 2 non-surgically, to domestic Welsh-type pony mares. After surgical transfer, 7 (77.8%) pregnancies were established and 4 foals were born. Twelve Grant's zebra embryos were transferred surgically to 5 pony and 7 domestic donkey recipients respectively and 1 non-surgically to a donkey; 3 (60%) zebra-in-horse pregnancies were established and 2 went to term. Only 2 (28.6%) zebra-in-donkey pregnancies were established but neither went to term, although one zebra foal was aborted alive at Day 292 but failed to survive. No pregnancies resulted from the non-surgical transfers. Measurement of chorionic gonadotrophin concentrations and parental-specific lymphocytotoxic antibodies in the serum of the recipient animals indicated a pronounced maternal immunological response to the extra-specific embryo, but this could not be correlated with success or failure of pregnancy. The results indicate that extra-specific embryo transfer may be a useful aid to breeding exotic equids in captivity.     

Co-transfer of parthenogenotes and single porcine embryos leads to full-term development of the embryos

It has long been known that several embryos are needed to establish and maintain pregnancy during early gestation in pigs. In this study, we assessed whether co-transfer of parthenogenotes with a single embryo was sufficient to maintain development of the embryo. Embryos were recovered at morula and early blastocyst stages from gilts that had been artificially inseminated. Parthenogenotes were produced from oocytes matured in vitro, activated by electrical stimulation, and then cultured for 110h. Those that had developed to morula- or blastocyst-like stages at 110h post-activation were transferred to recipient pigs either with or without morula or blastocyst stage embryos. Parthenogenotes that were transferred to recipients in the absence of embryos were viable up to 30 days post-activation and formed limb-buds; at 40 days, however, all were dead or degenerate. Among pigs that received both parthenogenotes and a single embryo, seven of nine (77.8%) delivered a normal piglet at full-term. This study therefore demonstrates that parthenogenotes can be used in place of embryos to establish pregnancy and promote development of a single co-transferred embryo. This method may be applied to rescue high value porcine embryos that are difficult to produce, such as transgenic cloned embryos, or for embryos frozen as a genetic resource.     

The current status of equine embryo transfer

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.     

Direct transfer of bovine embryos frozen-thawed in the presence of propylene glycol or ethylene glycol under on-farm conditions in an integrated embryo transfer program

An integrated bovine embryo transfer program was conducted in collaboration with 11 Japanese prefectural livestock experiment stations. The program was conducted to evaluate the practicability of the direct transfer method for bovine embryos frozen-thawed in the presence of propylene glycol (PG) or ethylene glycol (EG) under on-farm conditions. Embryos at the compacted morula to expanded blastocyst stages were collected from superovulated donors on Day 7 or 8 after estrus and equilibrated in 1.6 M PG or 1.8 M EG in Dulbecco's phosphate-buffered saline (DPBS) supplemented with 20% heat-inactivated calf serum. Embryos were then loaded individually into a 0.25-ml straw and placed directly into a cooling chamber of a programmable freezer precooled to -7 degrees C. After 2 min, the straw was seeded, maintained at -7 degrees C for 8 min more, and then cooled to -30 degrees C either at 0.3 degree C/min or 0.5 degree C/min before being plunged into liquid nitrogen. Embryos at the same stages were also frozen in the presence of 1.4 M glycerol (GLY) by a conventional method, which served as a control. The frozen embryos were thawed by allowing the straws to stand in air for 5 to 10 sec and then immersing them in a 30 degrees C water bath. Embryos frozen-thawed in the presence of PG or EG were nonsurgically transferred into the uterine horn without diluting the cryoprotectant. Embryos frozen-thawed in the presence of GLY were nonsurgically transferred after removing GLY either by the stepwise method (GLY-I) or by in situ dilution with 0.3 M sucrose solution (GLY-II). A total of 1,273 (PG: 400, EG: 418, GLY-I: 177, GLY-II; 278) frozen-thawed embryos was transferred into recipients, yielding 545 pregnancies (overall: 42.8%, PG: 36.0%, EG; 44.7%, GLY-I; 48.6%, GLY-II; 46.0%). The pregnancy rate with PG was significantly lower than that with EG or GLY-II (P < 0.05). The pregnancy rate was affected by the type of cryoprotectant, the region where the embryo transfer program was carried out, the developmental stage of the embryos, the parity of the recipients, and corpus luteum (CL) quality of the recipients. There were no differences in rates of abortion and stillbirth among the 3 cryoprotectants. The present study demonstrates that EG can be effectively used as a cryoprotectant for freezing and direct transfer of bovine embryos, and that the direct transfer method is applicable under on-farm conditions.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

First successful transfer of cryopreserved feline (Felis catus) embryos resulting in live offspring

Sexually mature domestic cats were hormonally stimulated to induce superovulation; embryo recovery was accomplished by laparotomy. Embryos were frozen by conventional embryo freezing methods used in the domestic cattle embryo transfer industry. Thawing was achieved in a 28 degrees C or 37 degrees C waterbath or in ambient air. Overnight culture of the frozen-thawed embryos in a supplemented Nutrient Mixture F-10 (Ham's) or Earle's Balanced Salt Solution with 20% heat-treated newborn calf serum resulted in five successful term litters from recipient queens. Embryo recipients who became pregnant had been treated with a subcutaneous injection of follicle-stimulating hormone (FSH-P) once every 24 hr for 6 days in the amount of 0.2 mg/day for the first 5 days and 0.1 mg on the sixth day, followed by two intramuscular 750 IU injections of human chorionic gonadotropin 24 hr apart, beginning on the same day as the sixth injection of FSH-P.     

Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida

The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET-DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates.     

Genetic diversity of Syrian Arabian horses

Although Arabian horses have been bred in strains for centuries and pedigrees have been recorded in studbooks, to date, little is known about the genetic diversity within and between these strains. In this study, we tested if the three main strains of Syrian Arabian horses descend from three founders as suggested by the studbook. We examined 48 horses representing Saglawi (n = 18), Kahlawi (n = 16) and Hamdani (n = 14) strains using the Equine SNP70K BeadChip. For comparison, an additional 24 Arabian horses from the USA and three Przewalski's horses as an out group were added. Observed heterozygosis (H_o) ranged between 0.30 and 0.32, expected heterozygosity (H_e) between 0.30 and 0.31 and inbreeding coefficients (F_is) between ?0.02 and ?0.05, indicating high genetic diversity within Syrian strains. Likewise, the genetic differentiation between the three Syrian strains was very low (F_st < 0.05). Hierarchical clustering showed a clear distinction between Arabian and Przewalski's horses. Among Arabian horses, we found three clusters containing either horses from the USA or horses from Syria or horses from Syria and the USA together. Individuals from the same Syrian Arabian horse strain were spread across different sub‐clusters. When analyzing Syrian Arabian horses alone, the best population differentiation was found with three distinct clusters. In contrast to expectations from the studbook, these clusters did not coincide with strain affiliation. Although this finding supports the hypothesis of three founders, the genetic information is not consistent with the currently used strain designation system. The information can be used to reconsider the current breeding practice. Beyond that, Syrian Arabian horses are an important reservoir for genetic diversity.     

Development and viability of frozen-thawed cattle embryos

Embryos recovered 7 to 8 days after estrus were frozen from -7 to -30 degrees C at 0.3 degrees C/min, from -30 to -33 degrees C at 0.1 degrees C/min, and then plunged into liquid nitrogen. They were thawed in a 25 degrees C waterbath. In a preliminary study, 15 of 18 embryos continued to develop during the 24-hour culture post-thaw in either Ham's F-10 or modified Dulbecco's phosphate buffered saline (PBS). In the main study, 5 of 20 embryos developed to 60-day pregnancies when embryos were transferred within 5 hours after thawing. The incidence of extended estrous cycles (pregnancy or presumed embryonic mortality) was 10 of 14, when the zona pellucida was intact after thawing, and 0 of 6, when it was ruptured or absent (P<.05). Embryos cultured in PBS tended to develop more readily than those in Ham's F-10 (15 of 20 vs 9 of 20, respectively, P reverse similar.1). Quality of the embryos, at recovery from the donor and after thawing, affected development in culture (19 of 27 embryos excellent at recovery developed vs 5 of 13 poor to very good, P reverse similar.1; 23 of 33 embryos good to excellent after thawing developed vs 1 of 7 poor, P<.05). The proportion of pyknotic nuclei in embryos which were cultured ranged from 18 to 100%. The pregnancy rate from embryos which were cultured was low (2 of 20). Thirty percent of frozen and thawed embryos had damaged zonae pellucidae. The study showed that: the pregnancy rate from frozen embryos was approximately half that achieved with unfrozen embryos; culturing embryos for 24 hours before transfer was not beneficial; the PBS culture system appears to be the system of choice for assessing embryo viability in vitro .     

Cryopreservation of wild mouse spermatozoa

Spermatozoa of wild mice from China, Czechoslovakia, Denmark, India, Japan and Switzerland were frozen and stored at -196 degrees C. After thawing, intact oocytes were inseminated in vitro with relatively high motility frozen-thawed mouse spermatozoa from Czechoslovakia, Denmark and India, while oocytes with a partially dissected zona were inseminated with low motility frozen-thawed spermatozoa from China, Japan and Switzerland. Embryos developing to the 2-cell stage from oocytes fertilized with frozen-thawed spermatozoa were transferred to the oviducts of female recipients on the first day of pseudopregnancy (day when a vaginal plug was confirmed). Successful embryo development to the 2-cell stage was 46 to 67%. Offspring resulted from 17 to 51% of these transferred 2-cell embryos.     

Molecular phylogeny and evolution of the Perissodactyla

The evolution of perissodactyls (rhinoceroses, tapirs, and horses) has been well studied primarily because of their extensive fossil record. Nevertheless, controversy persists regarding relationships of some of the extant taxa, reflecting inconsistencies between molecular and morphological studies. Here we examine the phylogenetic relationships of 16 living perissodactyl species by concatenating two mitochondrial and nine nuclear genes, and we estimate their divergence times using a relaxed Bayesian molecular clock approach. Our analyses recovered the monophyly of the suborders Ceratomorpha and Hippomorpha, and the families Rhinoceratidae, Tapiridae, and Equidae. We supported the early divergence of the Indian rhinoceros in the late Oligocene (26 Mya) relative to the Sumatran and African rhinoceroses, and the split of caballine (domestic horse and Przewalski's wild horse) and noncaballine equids (zebras and African and Asiatic asses) in the Pliocene (4 Mya). An important implication of this study is that Equus asinus, the African wild ass was found to be the sister taxon of Asiatic asses and zebras, diverging from the common ancestor with caballine horses 2 Mya. Rates of chromosome rearrangements were also evaluated in perissodactyls, placing a notably high rate of variation amongst equids, particularly within the zebra clade. The robust phylogenetic results presented here are relevant in terms of understanding the evolutionary history of this highly threatened group of mammals. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163 , 1289–1303.