Viability of equine embryos after puncture of the capsule and biopsy for preimplantation genetic diagnosis.

The equine embryo possesses a capsule that is considered essential for its survival. We assessed viability after breaching the capsule of early (Day 6) and expanded (Day 7 and 8) equine blastocysts by micromanipulation. The capsule was penetrated using a Piezo drill, and trophoblast biopsy samples were obtained for genetic analysis. Pregnancy rates for Day-6 embryos, which had intact zonae pellucidae at the time of recovery, were 3/3 for those biopsied immediately after recovery and 2/3 for those biopsied after being shipped overnight under warm (∼28 °C) conditions. The pregnancy rates for encapsulated Day-7 expanded blastocysts were 5/6 for those biopsied immediately and 5/6 for those biopsied after being shipped overnight warm. Two of four encapsulated Day-8 blastocysts, 790 and 1350 µm in diameter, established normal pregnancies after biopsy. Nine mares were allowed to maintain pregnancy, and they gave birth to nine normal foals. Biopsied cells from eight embryos that produced foals were subjected to whole-genome amplification. Sex was successfully determined from amplified DNA in 8/8 embryos. Identification of disease-causing mutations matched in the analyses of 6/6 samples for the sodium channel, voltage-gated, type IV, alpha subunit (SCN4A) gene and in 6/7 samples for the peptidylprolyl isomerase B (PPIB) gene, in embryo-foal pairs. Thus, the capsule of the equine embryo can be breached without impairing viability. Further work is needed to determine whether this breach is transient or permanent. These findings are relevant to the understanding of equine embryo development and to the establishment of methods for micromanipulation and embryo cryopreservation in this species.

A 4,103 marker integrated physical and comparative map of the horse genome.

A comprehensive second-generation whole genome radiation hybrid (RH II), cytogenetic and comparative map of the horse genome (2n = 64) has been developed using the 5000rad horse x hamster radiation hybrid panel and fluorescence in situ hybridization (FISH). The map contains 4,103 markers (3,816 RH; 1,144 FISH) assigned to all 31 pairs of autosomes and the X chromosome. The RH maps of individual chromosomes are anchored and oriented using 857 cytogenetic markers. The overall resolution of the map is one marker per 775 kilobase pairs (kb), which represents a more than five-fold improvement over the first-generation map. The RH II incorporates 920 markers shared jointly with the two recently reported meiotic maps. Consequently the two maps were aligned with the RH II maps of individual autosomes and the X chromosome. Additionally, a comparative map of the horse genome was generated by connecting 1,904 loci on the horse map with genome sequences available for eight diverse vertebrates to highlight regions of evolutionarily conserved syntenies, linkages, and chromosomal breakpoints. The integrated map thus obtained presents the most comprehensive information on the physical and comparative organization of the equine genome and will assist future assemblies of whole genome BAC fingerprint maps and the genome sequence. It will also serve as a tool to identify genes governing health, disease and performance traits in horses and assist us in understanding the evolution of the equine genome in relation to other species.

A BAC contig map over the proximal approximately 3.3 Mb region of horse chromosome 21.

A total of 207 BAC clones containing 155 loci were isolated and arranged into a map of linearly ordered overlapping clones over the proximal part of horse chromosome 21 (ECA21), which corresponds to the proximal half of the short arm of human chromosome 19 (HSA19p) and part of HSA5. The clones form two contigs - each corresponding to the respective human chromosomes - that are estimated to be separated by a gap of approximately 200 kb. Of the 155 markers present in the two contigs, 141 (33 genes and 108 STS) were generated and mapped in this study. The BACs provide a 4-5x coverage of the region and span an estimated length of approximately 3.3 Mb. The region presently contains one mapped marker per 22 kb on average, which represents a major improvement over the previous resolution of one marker per 380 kb obtained through the generation of a dense RH map for this segment. Dual color fluorescence in situ hybridization on metaphase and interphase chromosomes verified the relative order of some of the BACs and helped to orient them accurately in the contigs. Despite having similar gene order and content, the equine region covered by the contigs appears to be distinctly smaller than the corresponding region in human (3.3 Mb vs. 5.5-6 Mb) because the latter harbors a host of repetitive elements and gene families unique to humans/primates. Considering limited representation of the region in the latest version of the horse whole genome sequence EquCab2, the dense map developed in this study will prove useful for the assembly and annotation of the sequence data on ECA21 and will be instrumental in rapid search and isolation of candidate genes for traits mapped to this region.