Trio-binning of a hinny refines the comparative organization of the horse and donkey X chromosomes and reveals novel species-specific features.

We generated single haplotype assemblies from a hinny hybrid which significantly improved the gapless contiguity for horse and donkey autosomal genomes and the X chromosomes. We added over 15 Mb of missing sequence to both X chromosomes, 60 Mb to donkey autosomes and corrected numerous errors in donkey and some in horse reference genomes. We resolved functionally important X-linked repeats: the DXZ4 macrosatellite and ampliconic Equine Testis Specific Transcript Y7 (ETSTY7). We pinpointed the location of the pseudoautosomal boundaries (PAB) and determined the size of the horse (1.8 Mb) and donkey (1.88 Mb) pseudoautosomal regions (PARs). We discovered distinct differences in horse and donkey PABs: a testis-expressed gene, XKR3Y, spans horse PAB with exons1-2 located in Y and exon3 in the X-Y PAR, whereas the donkey XKR3Y is Y-specific. DXZ4 had a similar ~ 8 kb monomer in both species with 10 copies in horse and 20 in donkey. We assigned hundreds of copies of ETSTY7, a sequence horizontally transferred from Parascaris and massively amplified in equids, to horse and donkey X chromosomes and three autosomes. The findings and products contribute to molecular studies of equid biology and advance research on X-linked conditions, sex chromosome regulation and evolution in equids.

An Autosomal Translocation 73,XY,t(12;20)(q11;q11) in an Infertile Male Llama (Lama glama) With Teratozoospermia.

Structural chromosome abnormalities, such as translocations and inversions occasionally occur in all livestock species and are typically associated with reproductive and developmental disorders. Curiously, only a few structural chromosome aberrations have been reported in camelids, and most involved sex chromosomes. This can be attributed to a high diploid number (2n = 74) and complex chromosome morphology, which makes unambiguous identification of camelid chromosomes difficult. Additionally, molecular tools for camelid cytogenetics are sparse and have become available only recently. Here we present a case report about an infertile male llama with teratozoospermia and abnormal chromosome number 2n = 73,XY. This llama carries an autosomal translocation of chromosomes 12 and 20, which is the likely cause of defective spermatogenesis and infertility in this individual. Our analysis underlines the power of molecular cytogenetics methods over conventional banding-based chromosome analysis for explicit identification of normal and aberrant chromosomes in camelid karyotypes. This is the first case of a translocation and the first autosomal aberration reported in any camelid species. It is proof of principle that, like in other mammalian species, structural chromosome abnormalities contribute to reproductive disorders in camelids.

Novel drug delivery systems for ocular therapy: With special reference to liposomal ocular delivery.

Delivery of drugs to eyes is a great challenge to researchers because of a number of barriers in the eye preventing the actual dose from reaching the site. A number of ophthalmic delivery systems have been developed in the past couple of years that are not only new but also safe and reliable and help to overcome all those barriers in the eye which are responsible for the very less bioavailability of drugs. In this review, we tried to focus on current research in ocular delivery of drug substances giving special emphasis to liposomal delivery system. A brief analysis of other novel ocular delivery systems, ocular physiology, and microbial sources of disease are also highlighted herein. We analyzed the various research findings for churning a general idea for novel ocular delivery system and its future use. The novel formulations may overcome the addressed problems of ophthalmic medication and comply with the quality assurance issues. The liposomal delivery is advantageous as they have the ability to entrap both hydrophobic and hydrophilic drugs and are suitable for delivery to both the anterior and posterior segment of the eye. Therefore, the use of this alternative approach is quite a necessity.

Horse Y chromosome assembly displays unique evolutionary features and putative stallion fertility genes.

Dynamic evolutionary processes and complex structure make the Y chromosome among the most diverse and least understood regions in mammalian genomes. Here, we present an annotated assembly of the male specific region of the horse Y chromosome (eMSY), representing the first comprehensive Y assembly in odd-toed ungulates. The eMSY comprises single-copy, equine specific multi-copy, PAR transposed, and novel ampliconic sequence classes. The eMSY gene density approaches that of autosomes with the highest number of retained X-Y gametologs recorded in eutherians, in addition to novel Y-born and transposed genes. Horse, donkey and mule testis RNAseq reveals several candidate genes for stallion fertility. A novel testis-expressed XY ampliconic sequence class, ETSTY7, is shared with the parasite Parascaris genome, providing evidence for eukaryotic horizontal transfer and inter-chromosomal mobility. Our study highlights the dynamic nature of the Y and provides a reference sequence for improved understanding of equine male development and fertility.

Genetic assessment of leech species from yak (Bos grunniens) in the tract of Northeast India.

Yak is an iconic symbol of Tibet and high altitudes of Northeast India. It is highly cherished for milk, meat, and skin. However, yaks suffer drastic change in milk production, weight loss, etc, when infested by parasites. Among them, infestation by leeches is a serious problem in the Himalayan belt of Northeast India. The parasite feeds on blood externally or from body orifices, like nasopharynx, oral, rectum, etc. But there has been limited data about the leech species infesting the yak in that region because of the difficulties in morphological identification due to plasticity of the body, changes in shape, and surface structure and thus, warrants for the molecular characterization of leech. In anticipation, this study would be influential in proper identification of leech species infesting yak track and also helpful in inventorying of leech species in Northeast India. Here, we investigated, through combined approach of molecular markers and morphological parameters for the identification of leech species infesting yak. The DNA sequences of COI barcode fragment, 18S and 28S rDNA, were analyzed for species identification. The generated sequences were subjected to similarity match in global database and analyzed further through Neighbour-Joining, K2P distance based as well as ML approach. Among the three markers, only COI was successful in delineating species whereas the 18S and 28S failed to delineate the species. Our study confirmed the presence of the species from genus Hirudinaria, Haemadipsa, Whitmania, and one species Myxobdella annandalae, which has not been previously reported from this region.

Copy number variation in the horse genome.

We constructed a 400K WG tiling oligoarray for the horse and applied it for the discovery of copy number variations (CNVs) in 38 normal horses of 16 diverse breeds, and the Przewalski horse. Probes on the array represented 18,763 autosomal and X-linked genes, and intergenic, sub-telomeric and chrY sequences. We identified 258 CNV regions (CNVRs) across all autosomes, chrX and chrUn, but not in chrY. CNVs comprised 1.3% of the horse genome with chr12 being most enriched. American Miniature horses had the highest and American Quarter Horses the lowest number of CNVs in relation to Thoroughbred reference. The Przewalski horse was similar to native ponies and draft breeds. The majority of CNVRs involved genes, while 20% were located in intergenic regions. Similar to previous studies in horses and other mammals, molecular functions of CNV-associated genes were predominantly in sensory perception, immunity and reproduction. The findings were integrated with previous studies to generate a composite genome-wide dataset of 1476 CNVRs. Of these, 301 CNVRs were shared between studies, while 1174 were novel and require further validation. Integrated data revealed that to date, 41 out of over 400 breeds of the domestic horse have been analyzed for CNVs, of which 11 new breeds were added in this study. Finally, the composite CNV dataset was applied in a pilot study for the discovery of CNVs in 6 horses with XY disorders of sexual development. A homozygous deletion involving AKR1C gene cluster in chr29 in two affected horses was considered possibly causative because of the known role of AKR1C genes in testicular androgen synthesis and sexual development. While the findings improve and integrate the knowledge of CNVs in horses, they also show that for effective discovery of variants of biomedical importance, more breeds and individuals need to be analyzed using comparable methodological approaches.

A nanovesicle topical formulation of Bhut Jolokia (hottest capsicum): a potential anti-arthritic medicine.

OBJECTIVES: Bhut Jolokia is a capsicum variety indigenous to Northeast India and is recognized as the hottest capsicum variety of the world. The ethnobotanical survey revealed that it has potential anti-arthritic activity but its topical adverse events restrict its usability. In the present study, the semipurified capsaicinoids extract of Bhut Jolokia was formulated as a topical formulation via ethosomal nanovesicle approach, which enhanced the acceptability. METHODS: Prepared formulation was optimized by surface response methodology and characterized for morphology, zeta potential, differential scanning calorimetry study and permeation and penetration studies. The experimental formulations were characterized on Freunds complete adjuvant-induced chronic arthritis model. RESULTS: Ethosomal nanovesicles prepared with the semipurified capsaicinoids extract demonstrated good anti-arthritic activity in rat model, superior to Thermagel (a marketed formulation of capsaicin) in the reduction of joint swelling and pain throughout the observation period. Nanovesicle formulation showed better tolerability and acceptance on both animal and human models. Results obtained from the study strengthen the potential application of Bhut Jolokia semipurified extract in an ethosomal nanovesicle carrier as a topical analgesic as well as an anti-arthritic. CONCLUSION: The significant positive results, with reduced irritant effect of the semipurified capsaicinoids extract of Bhut Jolokia-loaded ethosomal nanovesicle carrier, suggest that it could be one of the choices for formulation development in anti-arthritic medicine.

Ovarian dysgenesis in an alpaca with a minute chromosome 36.

A 4-year-old female alpaca (Lama pacos [LPA]) was presented to the Oregon State Veterinary Teaching Hospital for failure to display receptive behavior to males. Although no abnormalities were found on physical examination, transrectal ultrasonographic examination of the reproductive tract revealed uterine hypoplasia and ovarian dysgenesis. Cytogenetic analysis demonstrated a normal female 74,XX karyotype with 1 exceptionally small (minute) homologue of autosome LPA36. Chromosome analysis by Giemsa staining and DAPI- and C-banding revealed that the minute LPA36 was submetacentric, AT-rich, and largely heterochromatic. Because of the small size and lack of molecular markers, it was not possible to identify the origin of the minute. There is a need to improve molecular cytogenetic tools to further study the phenomenon of this minute chromosome and its relation to female reproduction in alpacas and llamas.

Development and application of camelid molecular cytogenetic tools.

Cytogenetic chromosome maps offer molecular tools for genome analysis and clinical cytogenetics and are of particular importance for species with difficult karyotypes, such as camelids (2n = 74). Building on the available human-camel zoo-fluorescence in situ hybridization (FISH) data, we developed the first cytogenetic map for the alpaca (Lama pacos, LPA) genome by isolating and identifying 151 alpaca bacterial artificial chromosome (BAC) clones corresponding to 44 specific genes. The genes were mapped by FISH to 31 alpaca autosomes and the sex chromosomes; 11 chromosomes had 2 markers, which were ordered by dual-color FISH. The STS gene mapped to Xpter/Ypter, demarcating the pseudoautosomal region, whereas no markers were assigned to chromosomes 14, 21, 22, 28, and 36. The chromosome-specific markers were applied in clinical cytogenetics to identify LPA20, the major histocompatibility complex (MHC)-carrying chromosome, as a part of an autosomal translocation in a sterile male llama (Lama glama, LGL; 2n = 73,XY). FISH with LPAX BACs and LPA36 paints, as well as comparative genomic hybridization, were also used to investigate the origin of the minute chromosome, an abnormally small LPA36 in infertile female alpacas. This collection of cytogenetically mapped markers represents a new tool for camelid clinical cytogenetics and has applications for the improvement of the alpaca genome map and sequence assembly.

A comprehensive whole-genome integrated cytogenetic map for the alpaca (Lama pacos).

Genome analysis of the alpaca (Lama pacos, LPA) has progressed slowly compared to other domestic species. Here, we report the development of the first comprehensive whole-genome integrated cytogenetic map for the alpaca using fluorescence in situ hybridization (FISH) and CHORI-246 BAC library clones. The map is comprised of 230 linearly ordered markers distributed among all 36 alpaca autosomes and the sex chromosomes. For the first time, markers were assigned to LPA14, 21, 22, 28, and 36. Additionally, 86 genes from 15 alpaca chromosomes were mapped in the dromedary camel (Camelus dromedarius, CDR), demonstrating exceptional synteny and linkage conservation between the 2 camelid genomes. Cytogenetic mapping of 191 protein-coding genes improved and refined the known Zoo-FISH homologies between camelids and humans: we discovered new homologous synteny blocks (HSBs) corresponding to HSA1-LPA/CDR11, HSA4-LPA/CDR31 and HSA7-LPA/CDR36, and revised the location of breakpoints for others. Overall, gene mapping was in good agreement with the Zoo-FISH and revealed remarkable evolutionary conservation of gene order within many human-camelid HSBs. Most importantly, 91 FISH-mapped markers effectively integrated the alpaca whole-genome sequence and the radiation hybrid maps with physical chromosomes, thus facilitating the improvement of the sequence assembly and the discovery of genes of biological importance.

Stallion sperm transcriptome comprises functionally coherent coding and regulatory RNAs as revealed by microarray analysis and RNA-seq.

Mature mammalian sperm contain a complex population of RNAs some of which might regulate spermatogenesis while others probably play a role in fertilization and early development. Due to this limited knowledge, the biological functions of sperm RNAs remain enigmatic. Here we report the first characterization of the global transcriptome of the sperm of fertile stallions. The findings improved understanding of the biological significance of sperm RNAs which in turn will allow the discovery of sperm-based biomarkers for stallion fertility. The stallion sperm transcriptome was interrogated by analyzing sperm and testes RNA on a 21,000-element equine whole-genome oligoarray and by RNA-seq. Microarray analysis revealed 6,761 transcripts in the sperm, of which 165 were sperm-enriched, and 155 were differentially expressed between the sperm and testes. Next, 70 million raw reads were generated by RNA-seq of which 50% could be aligned with the horse reference genome. A total of 19,257 sequence tags were mapped to all horse chromosomes and the mitochondrial genome. The highest density of mapped transcripts was in gene-rich ECA11, 12 and 13, and the lowest in gene-poor ECA9 and X; 7 gene transcripts originated from ECAY. Structural annotation aligned sperm transcripts with 4,504 known horse and/or human genes, rRNAs and 82 miRNAs, whereas 13,354 sequence tags remained anonymous. The data were aligned with selected equine gene models to identify additional exons and splice variants. Gene Ontology annotations showed that sperm transcripts were associated with molecular processes (chemoattractant-activated signal transduction, ion transport) and cellular components (membranes and vesicles) related to known sperm functions at fertilization, while some messenger and micro RNAs might be critical for early development. The findings suggest that the rich repertoire of coding and non-coding RNAs in stallion sperm is not a random remnant from spermatogenesis in testes but a selectively retained and functionally coherent collection of RNAs.

Genome-wide association study implicates testis-sperm specific FKBP6 as a susceptibility locus for impaired acrosome reaction in stallions.

Impaired acrosomal reaction (IAR) of sperm causes male subfertility in humans and animals. Despite compelling evidence about the genetic control over acrosome biogenesis and function, the genomics of IAR is as yet poorly understood, providing no molecular tools for diagnostics. Here we conducted Equine SNP50 Beadchip genotyping and GWAS using 7 IAR-affected and 37 control Thoroughbred stallions. A significant (P<6.75E-08) genotype-phenotype association was found in horse chromosome 13 in FK506 binding protein 6 (FKBP6). The gene belongs to the immunophilins FKBP family known to be involved in meiosis, calcium homeostasis, clathrin-coated vesicles, and membrane fusions. Direct sequencing of FKBP6 exons in cases and controls identified SNPs g.11040315G>A and g.11040379C>A (p.166H>N) in exon 4 that were significantly associated with the IAR phenotype both in the GWAS cohort (n = 44) and in a large multi-breed cohort of 265 horses. All IAR stallions were homozygous for the A-alleles, while this genotype was found only in 2% of controls. The equine FKBP6 was exclusively expressed in testis and sperm and had 5 different transcripts, of which 4 were novel. The expression of this gene in AC/AG heterozygous controls was monoallelic, and we observed a tendency for FKBP6 up-regulation in IAR stallions compared to controls. Because exon 4 SNPs had no effect on the protein structure, it is likely that FKBP6 relates to the IAR phenotype via regulatory or modifying functions. In conclusion, FKBP6 was considered a susceptibility gene of incomplete penetrance for IAR in stallions and a candidate gene for male subfertility in mammals. FKBP6 genotyping is recommended for the detection of IAR-susceptible individuals among potential breeding stallions. Successful use of sperm as a source of DNA and RNA propagates non-invasive sample procurement for fertility genomics in animals and humans.

Total RNA isolation from stallion sperm and testis biopsies.

Sperm mRNA transcriptional profiles can be used to evaluate male fertility, yet differences between species in sperm attributes and packaging require adjustments in sperm RNA isolation protocols. The objective was to optimize RNA isolation methodology for fresh, frozen, and extended ejaculates, and epididymal sperm of stallions. Additionally, a protocol for RNA isolation from testis biopsies was established. Separation of sperm from somatic cells was critical for assuring the isolation of sperm-specific RNA. The highest purity was obtained by centrifuging ejaculates and epididymal sperm at 200 x g for 30 min through a 40% Equipure silanized silica particle solution. Sperm RNA isolation was more efficient with TRIzol reagent than with a spin-column based method; it resulted in 2 microg of total RNA per 100 x 10(6) sperm. To evaluate RNA quantity and quality, we used a NanoDrop spectrophotometer and Agilent Bioanalyzer. A protocol for reverse transcriptase PCR with equine primers for PRM2 and PTPRC genes was developed to determine sperm RNA contamination with genomic DNA or RNA from somatic cells. By these methods, hybridization- and sequencing-quality RNA was isolated from 11 samples of stallion sperm. Stallion testis biopsy with a 14 gauge 22 mm deep biopsy needle yielded approximately 12 microg of good quality total RNA, and could serve as an alternative to excision surgery for sample procurement. Compared to RNA isolation from testis, the sperm required advanced processing and RNA quality control. The described methodologies provided a foundation to establish functional genomic studies of stallion fertility.

Blood chimerism confounds genetic relative susceptibility testing for classical scrapie in sheep.

Classical scrapie disease is a transmissible spongiform encephalopathy of sheep that is enzootic in the United States. Susceptibility of sheep to classical scrapie is linked to single nucleotide polymorphisms in the prion protein gene (PRNP), forming the basis for genetic testing strategies used by national efforts to eradicate scrapie. Such efforts are occasionally hampered by inconclusive results stemming from the detection of "complex" genotypes. Naturally occurring cases of ovine chimerism are thought to account for some of these instances. In the current report, 4 naturally occurring ovine chimeras are documented through cytogenetic and molecular analyses. All 4 of these sheep had chimeric cells circulating in their blood. Blood and alternate tissue samples of ear punch and hair bulbs from one of these chimeras was submitted in batch with similar samples from control sheep for routine scrapie genetic relative susceptibility testing. A complex PRNP genotype was detected in the blood of the chimeric female but not in the alternate tissue samples or in the control sheep samples. The results demonstrate that naturally occurring blood chimerism can confound current testing efforts. The potential impacts of undetected chimeras on current scrapie eradication efforts are discussed.