Topic:Cloning
Cloning in horses involves the process of creating a genetically identical copy of an original horse through somatic cell nuclear transfer (SCNT). This technique involves transferring the nucleus of a somatic cell from the donor horse into an enucleated oocyte, which is then stimulated to develop into an embryo and implanted into a surrogate mare. Cloning has been utilized for various purposes, including the preservation of valuable genetics, reproduction of geldings, and research into genetic diseases. The practice raises discussions on genetic diversity, animal welfare, and ethical considerations. This page compiles peer-reviewed research studies and scholarly articles that explore the methodologies, applications, and implications of cloning in equine science.
The genome of Przewalski’s horse ( Equus ferus przewalskii ). The Przewalski's horse ( ) is an endangered equid native to the steppes of central Asia. After becoming extinct in the wild, multiple conservation efforts convened to preserve the species including captive breeding programs, reintroduction and monitoring systems, protected lands, and cloning. Availability of a highly contiguous reference genome is essential to support these continued efforts. We used Oxford Nanopore sequencing to produce a scaffold-level 2.5 Gb nuclear assembly and 16,002 bp mitogenome from a captive Przewalski's mare. All assembly drafts were generated from 111 Gb of sequenc...
Multiplex Detection of Transgenes Using πCode Technology for Gene Doping Control. To ensure fair competition and sports integrity, gene doping is prohibited in horseracing and equine sports. One gene doping method is by administering exogenous genes, called transgenes, to postnatal animals. Although several transgene detection methods have been developed for horses, many are unsuitable for multiplex detection. In this proof-of-concept study, we developed a highly sensitive and multiplex transgene detection method using multiple πCode with identification patterns printed on the surface. The following steps were employed: (1) multiplex polymerase chain reaction amplification...
Horse Somatic Cell Nuclear Transfer Using Zona Pellucida-Enclosed and Zona-Free Oocytes. Horse cloning by somatic cell nuclear transfer (SCNT) is an attractive scientific and commercial endeavor. Moreover, SCNT allows generating genetically identical animals from elite, aged, castrated, or deceased equine donors. Several variations in the horse SCNT method have been described, which may be useful for specific applications. This chapter describes a detailed protocol for horse cloning, thus including SCNT protocols using zona pellucida (ZP)-enclosed or ZP-free oocytes for enucleation. These SCNT protocols are under routine use for commercial equine cloning.
Cloning horses by somatic cell nuclear transfer: Effects of oocyte source on development to foaling. The cloning of horses is a commercial reality, yet the availability of oocytes for cloned embryo production remains a major limitation. Immature oocytes collected from abattoir-sourced ovaries or from live mares by ovum pick-up (OPU) have both been used to generate cloned foals. However, the reported cloning efficiencies are difficult to compare due to the different somatic cell nuclear transfer (SCNT) techniques and conditions used. The objective of this retrospective study was to compare the in vitro and in vivo development of equine SCNT embryos produced using oocytes recovered from abatt...
The Amelioration of Grazing through Physiological Integration by a Clonal Dune Plant. Rhizomatous growth and associated physiological integration can allow a clonal dune species to potentially compensate for the selective removal of leaves associated with herbivory. is a rhizomatous clonal plant species that is abundant in the coastal dune environments of the southeastern United States that are inhabited by large feral horse populations. has been shown to integrate resources among ramets within extensive clones as an adaptation to resource heterogeneity in sandy soils. In this study, we hypothesized that clonal integration is a mechanism that promotes persistence in these co...
First sex modification case in equine cloning. Somatic cell nuclear transfer (SCNT) is an asexual reproductive technique where cloned offspring contain the same genetic material as the original donor. Although this technique preserves the sex of the original animal, the birth of sex-reversed offspring has been reported in some species. Here, we report for the first time the birth of a female foal generated by SCNT of a male nuclear donor. After a single SCNT procedure, 16 blastocysts were obtained and transferred to eight recipient mares, resulting in the birth of two clones: one male and one female. Both animals had identical genetic prof...
Exploiting V-Gene Bias for Rapid, High-Throughput Monoclonal Antibody Isolation from Horses. Horses and humans share a close relationship that includes both species' viromes. Many emerging infectious diseases can be transmitted between horses and humans and can exhibit mortality rates as high as 90% in both populations. Antibody biologics represents an emerging field of rapidly discoverable and potent antiviral therapeutics. These biologics can be used to provide passive immunity, as well as blueprints for the rational design of novel active vaccine antigens. Here, we exploit the limited diversity of immunoglobulin variable genes used by horses to develop a rapid, high-throughput mono...
Detection of Indiscriminate Genetic Manipulation in Thoroughbred Racehorses by Targeted Resequencing for Gene-Doping Control. The creation of genetically modified horses is prohibited in horse racing as it falls under the banner of gene doping. In this study, we developed a test to detect gene editing based on amplicon sequencing using next-generation sequencing (NGS). We designed 1012 amplicons to target 52 genes (481 exons) and 147 single-nucleotide variants (SNVs). NGS analyses showed that 97.7% of the targeted exons were sequenced to sufficient coverage (depth > 50) for calling variants. The targets of artificial editing were defined as homozygous alternative (HomoALT) and compound heterozygous alternative (ALT1/...
Use of recombinant calflagin protein as a potential candidate for diagnosis of Trypanosoma evansi infection. Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies a...
Use of mitochondrial sequencing to detect gene doping in horses via gene editing and somatic cell nuclear transfer. Gene editing and subsequent cloning techniques offer great potential not only in genetic disease correction in domestic animals but also in livestock production by enhancement of desirable traits. The existence of the technology, however, leaves it open to potential misuse in performance-led sports such as horseracing and other equestrian events. Recent advances in equine gene editing, regarding the generation of gene-edited embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer, have highlighted the need to develop tools to detect potential prohibited use of the technology. On...
Genetic analysis of mitochondrial DNA from ancient Equus caballus bones found at archaeological site of Joseon dynasty period capital area. To understand the domestication and spread of horses in history, genetic information is essential. However, mitogenetic traits of ancient or medieval horses have yet to be comprehensively revealed, especially for East Asia. This study thus set out to reveal the maternal lineage of skeletal horse remains retrieved from a 15th century archaeological site (Gongpyeongdong) at Old Seoul City in South Korea. Methods: We extracted DNA from the femur of Equus caballus (SNU-A001) from Joseon period Gongpyeongdong site. Mitochondrial (mt) DNA (HRS 15128-16116) of E. caballus was amplified by polymerase ...
The Induced Expression of BPV E4 Gene in Equine Adult Dermal Fibroblast Cells as a Potential Model of Skin Sarcoid-like Neoplasia. The equine sarcoid is one of the most common neoplasias in the family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that - and -mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like...
Genetic Manipulation of the Equine Oocyte and Embryo. As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in t...
Two Novel Cases of Autosomal Translocations in the Horse: Warmblood Family Segregating t(4;30) and a Cloned Arabian with a de novo t(12;25). We report 2 novel autosomal translocations in the horse. In Case 1, a breeding stallion with a balanced t(4p;30) had produced normal foals and those with congenital abnormalities. Of his 9 phenotypically normal offspring, 4 had normal karyotypes, 4 had balanced t(4p;30), and 1 carried an unbalanced translocation with tertiary trisomy of 4p. We argue that unbalanced forms of t(4p;30) are more tolerated and result in viable congenital abnormalities, without causing embryonic death like all other known equine autosomal translocations. In Case 2, two stallions produced by somatic cell nuclear tran...
Generation of myostatin edited horse embryos using CRISPR/Cas9 technology and somatic cell nuclear transfer. The application of new technologies for gene editing in horses may allow the generation of improved sportive individuals. Here, we aimed to knock out the myostatin gene (MSTN), a negative regulator of muscle mass development, using CRISPR/Cas9 and to generate edited embryos for the first time in horses. We nucleofected horse fetal fibroblasts with 1, 2 or 5 µg of 2 different gRNA/Cas9 plasmids targeting the first exon of MSTN. We observed that increasing plasmid concentrations improved mutation efficiency. The average efficiency was 63.6% for gRNA1 (14/22 edited clonal cell lines) and 96.2% ...
Horse ooplasm supports in vitro preimplantation development of zebra ICSI and SCNT embryos without compromising YAP1 and SOX2 expression pattern. Several equids have gone extinct and many extant equids are currently considered vulnerable to critically endangered. This work aimed to evaluate whether domestic horse oocytes support preimplantation development of zebra embryos obtained by intracytoplasmic sperm injection (ICSI, zebroid) and cloning, and to study the Hippo signaling pathway during the lineage specification of trophectoderm cells and inner cell mass cells. We first showed that zebra and horse sperm cells induce porcine oocyte activation and recruit maternal SMARCA4 during pronuclear formation. SMARCA4 recruitment showed to be...
Metagenomic sequencing of clinical samples reveals a single widespread clone of Lawsonia intracellularis responsible for porcine proliferative enteropathy. Lawsonia intracellularis is a Gram-negative obligate intracellular bacterium that is the aetiological agent of proliferative enteropathy (PE), a common intestinal disease of major economic importance in pigs and other animal species. To date, progress in understanding the biology of L. intracellularis for improved disease control has been hampered by the inability to culture the organism in vitro. In particular, our understanding of the genomic diversity and population structure of clinical L. intercellularis is very limited. Here, we utilized a metagenomic shotgun approach to directly sequenc...
Assessing the fertility of two mares cloned from the same founder animal. Two cloned mares, produced from the same sample of skin fibroblasts, were bred during four breeding seasons from their second year of age, as embryo donors, in exactly the same conditions, using the same stallions for both cloned mares. The aim of this study was to test the embryo donor potential of cloned mares and to compare the results obtained from two cloned mares of the same mare with other embryo donor mares (n = 31-39 per breeding season) at the same stud. For both cloned mares, 19 embryos were recovered by 43 collection attempts (44%) (7/22 for one; 12/21 for the other), 16 (84%) pr...
Targeting epigenetic nuclear reprogramming in aggregated cloned equine embryos. Epigenetic perturbations during the reprogramming process have been described as the primary cause of the low efficiency of somatic cell nuclear transfer (SCNT). In this study, we tested three strategies targeting nuclear reprogramming to investigate effects on equine SCNT. First, we evaluated the effect of treating somatic cells with chetomin, a fungal secondary metabolite reported to inhibit the trimethylation on histone 3 lysine 9 (H3K9 me3). Second, caffeine was added to the culture medium during the enucleation of oocytes and before activation of reconstructed embryos as a protein phospha...
Resiliency of equid H19 imprint to somatic cell reprogramming by oocyte nuclear transfer and genetically induced pluripotency†. Cell reprogramming by somatic cell nuclear transfer and in induced pluripotent stem cells is associated with epigenetic modifications that are often incompatible with embryonic development and differentiation. For instance, aberrant DNA methylation patterns of the differentially methylated region and biallelic expression of H19-/IGF2-imprinted gene locus have been associated with abnormal growth of fetuses and placenta in several mammalian species. However, cloned horses are born with normal sizes and with no apparent placental anomalies, suggesting that H19/IGF2 imprinting may be epigenetical...
Approach to Strain Selection and the Propagation of Viral Stocks for Venezuelan Equine Encephalitis Virus Vaccine Efficacy Testing under the Animal Rule. Licensure of a vaccine to protect against aerosolized Venezuelan equine encephalitis virus (VEEV) requires use of the U.S. Food and Drug Administration (FDA) Animal Rule to assess vaccine efficacy as human studies are not feasible or ethical. An approach to selecting VEEV challenge strains for use under the Animal Rule was developed, taking into account Department of Defense (DOD) vaccine requirements, FDA Animal Rule guidelines, strain availability, and lessons learned from the generation of filovirus challenge agents within the Filovirus Animal Nonclinical Group (FANG). Initial down-selectio...
Enhancing Fertility in Mares: Recombinant Equine Gonadotropins. Advanced reproductive technologies have been developed to enhance fertility in mares and stallions. Some of these technologies in mares include superovulation, embryo transfer (ET), intracytoplasmic sperm injection (ICSI), oocyte transfer (OT), gamete intrafallopian transfer (GIFT), and cloning. Superovulation can provide multiple oocytes for these techniques. This review will focus on how recombinant equine follicle-stimulating hormone (reFSH) and recombinant equine luteinizing hormone (reLH) are important for superovulation and ET and may be useful for ICSI, OT, GIFT, and cloning. Superovula...
Development of the central nervous system in equine twin fetuses derived by somatic cell nuclear transfer. Because of the growing importance of horses in leisure and several sports, somatic cell nuclear transfer (SCNT) is being used more frequently for cloning animals for performance and reproductive purposes. However, because of the need to perforate the zona pellucida during microsurgical reconstruction of the oocyte, it is possible that SCNT-derived embryos undergo premature hatching, resulting in embryo bisection and twinning. Therefore, because equine twin pregnancies often lead to abnormal embryo development and pregnancy failure, we performed a detailed comparative assessment of equine twin ...
Anaerobic fungal communities differ along the horse digestive tract. Anaerobic fungi are potent fibre degrading microbes in the equine hindgut, yet our understanding of their diversity and community structure is limited to date. In this preliminary work, using a clone library approach we studied the diversity of anaerobic fungi along six segments of the horse hindgut: caecum, right ventral colon (RVC), left ventral colon (LVC), left dorsal colon (LDC), right dorsal colon (RDC) and rectum. Of the 647 ITS1 clones, 61.7 % were assigned to genus level groups that are so far without any cultured representatives, and 38.0 % were assigned to the cultivated genera Ne...
Cloning and Expression of Immunogenic Regions of EMA-1 Gene of Theileria equi From Infected Horses. Diversity among the pathogenic strains of Theileria equi (T. equi), a major agent of equine piroplasmosis, can affect the appropriate detection of parasite and host immunization. Production of recombinant surface proteins from an infected horse in natural endemic area provides a reliable tool for immunodiagnosis of parasite. Regarding this, the present study was targeted toward the cloning, expression, and purification of the immunogenic regions of equine merozoite antigen 1 (EMA-1 gene), as one of the most important immunodominant surface proteins in T. equi, from naturally infected horses in...
Assisted reproductive techniques in mares. A wide variety of assisted reproductive techniques (ARTs) are available to aid in managing aspects of equine reproduction. Embryo recovery and transfer can be used to obtain more than one foal per mare per year, and to obtain foals from mares that cannot carry a foal to term. Oocyte recovery and either transfer to the oviduct of an inseminated recipient mare (oocyte transfer), or intracytoplasmic sperm injection (ICSI) and embryo culture can be used to obtain foals from mares with some types of subfertility, such as problems of the tubular tract. ICSI can be used to obtain foals when sperm num...
The development of in vitro embryo production in the horse. The development of techniques to produce equine embryos in vitro is reviewed with specific reference to intracytoplasmic sperm injection (ICSI). Unexplored 50 years ago, this technology has progressed rapidly in the last 20 years to become a commercial reality for the equine breeding industry. Improvements in our understanding of oocyte and embryo competence in the horse have been key factors in overcoming some of the initial problems associated with ICSI. It is now possible to obtain high nuclear maturation and cleavage rates in vitro and the most limiting factor, presently, is the low rate o...
Culture of somatic cells isolated from frozen-thawed equine semen using fluorescence-assisted cell sorting. Nuclear transfer using somatic cells from frozen semen (FzSC) would allow cloning of animals for which no other genetic material is available. Horses are one of the few species for which cloning is commercially feasible; despite this, there is no information available on the culture of equine FzSC. After preliminary trials on equine FzSC, recovered by density-gradient centrifugation, resulted in no growth, we hypothesized that sperm in the culture system negatively affected cell proliferation. Therefore, we evaluated culture of FzSC isolated using fluorescence-assisted cell sorting. In Exp. 1,...