Analysis of morphological disorders and ploidy in domestic cat blastocysts.

This study describes, for the first time, the relationship between morphology and ploidy in domestic cat embryos. Blastocyst morphology and quality were assessed using time-lapse recordings, while ploidy was analyzed using fluorescence in situ hybridization. Out of 54 blastocysts, clear fluorescence signals for all the molecular probes used were observed in 24 (44.4%) blastocysts, while in another 14 (25.9%) blastocysts, fluorescence signals only allowed for sex assessment. No clear signals were observed in the remaining 16 blastocysts (29.7%). Of the 24 blastocysts with clear signals, normal ploidy was detected in 10 (41.4%), 7 (29.2%) were diagnosed as haploid, and the remaining 7 blastocysts (29.2%) were mosaics. Additionally, results showed the distribution of diploid, haploid, and mosaic blastocysts in relation to the occurrence of morphological disorders and to embryo quality. The presence of abnormal embryo morphology and karyotype disorders may affect further development and the pregnancy rate. Due to the comparable proportion of good and poor quality blastocysts with disturbed ploidy, it is important to implement new methods of embryo assessment, especially when techniques used in humans, such as pronuclear observation, cannot be used.

Sperm chromosome segregation of rob(4;16) and rob(4;16)inv(4) in the brown brocket deer (Mazama gouazoubira).

The genus Mazama stands out among the Neotropical deer due to their wide intra and interspecific karyotypic diversification, which is associated with an accentuated chromosomal fragility. There are reports of heterozygous Robertsonian translocation (RT) carriers in a free-range population of Mazama gouazoubira (brown brocket deer), as well as in captive animals of this and other species of the genus. To analyze possible negative impacts of heterozygous chromosome rearrangements on reproductive fitness of the carriers, we performed an analysis of sperm meiotic segregation in four brown brocket bucks, carriers of a rob(4;16), and compared the results with those of a normal buck. We established a reliable FISH and sperm-FISH protocol for the brown brocket deer using bovine (Bos taurus; diploid number, 2n = 60) whole chromosome painting (WCP) and BAC probes. Using BAC probes, we revealed the presence of a paracentric inversion (PAI) of the fused chromosome 4 in two of the four analyzed RT carriers. The mean frequency of normal/balanced sperm in the translocation carriers was significantly lower than in the normal buck (94.78% vs 98.40%). The mean value of total unbalanced spermatozoa was almost doubled in the RT/PAI carriers (6.68%) when compared to RT carriers (3.76%), but the difference was not statistically significant. This study demonstrated the efficiency of FISH with bovine WCP and BAC probes in the characterization of chromosome rearrangements and gametic segregation patterns in brown brocket deer. Our results indicate a low to moderate increase in the rates of unbalanced meiotic segregation products in brown brocket bucks heterozygous for RT and RT/PAIs.

Correlation between integration of high-risk HPV genome into human DNA detected by molecular combing and the severity of cervical lesions: first results of the EXPL-HPV-002 study.

OBJECTIVES: The aim of the EXPL-HPV-002 study is to evaluate the integration of 14 high-risk HPV as a biomarker of the severity and the progression of cervical lesions. Such a „triage biomarker“ would help to reduce the number of unnecessary colposcopies, to avoid over-treatment of lesions that spontaneously regress and to better target the lesions requiring treatment. DESIGN: EXPL-HPV-002 is a prospective, open-label, single arm, GCP study conducted at 2 clinical sites in the Czech Republic. SETTINGS: Investigations centers: Private Gynecology Center, Brno; Gynecological and Obstetrical Clinic, Brno; Genotyping central lab: NRL for Papillomaviruses and polyomaviruses, IHBT, Prague; Histology Central reading: Aeskulab Pathology, Prague; Molecular combing HPV test: Genomic Vision, Bagneux. METHODS: From June 2016 to May 2018, 688 patients aged 25-65, referred to colposcopy after an abnormal Pap-smear, were enrolled in the study. Among them 60% were found HPV high-risk. The study is divided in two phases: 1. a cross-sectional phase using data collected at first visit (colposcopy images ± histology, pap-smear for HPV genotyping and molecular combing) to study the association between HPV integration status versus colposcopy and histology grades; 2. a longitudinal phase using data collected in follow-up visits: cytology at 6, 18 and 30 months and colposcopy ± histology at 12, 24 and 36 months. A pap-smear collected at 12, 24 and 36 months allows to perform genotyping and molecular combing. HPV integration status is analyzed in comparison with the evolution of lesions, viral clearance and HPV genotype. HPV genotyping and molecular combing were performed on pap-smear samples in central laboratories. Histology data were reviewed by central reading. RESULTS: The transversal phase of the study is achieved and shows that the HPV integration into the human DNA, monitored by molecular combing, can significantly differentiate normal subjects from women with cervical lesions or cancer. CONCLUSION: HPV integration into the host genome, monitored by Genomic Visions technology, is a reliable diagnostic biomarker that will greatly help clinicians to improve their medical decision tree.

The internal structure of magnetic nanoparticles determines the magnetic response.

This work aims to emphasize that the magnetic response of single-domain magnetic nanoparticles (NPs) is driven by the NPs' internal structure, and the NP size dependencies of magnetic properties are overestimated. The relationship between the degree of the NPs' crystallinity and magnetic response is unambiguously demonstrated in eight samples of uniform maghemite/magnetite NPs and corroborated with the results obtained for about 20 samples of spinel ferrite NPs with different degrees of crystallinity. The NP samples were prepared by the thermal decomposition of an organic iron precursor subjected to varying reaction conditions, yielding variations in the NP size, shape and relative crystallinity. We characterized the samples by using several complementary methods, such as powder X-ray diffraction (PXRD), transmission electron microscopy (TEM), high resolution TEM (HR-TEM) and Mössbauer spectroscopy (MS). We evaluated the NPs' relative crystallinity by comparing the NP sizes determined from TEM and PXRD and further inspecting the NPs' internal structure and relative crystallinity by using HR-TEM. The results of the structural characterization were put in the context of the NPs' magnetic response. In this work, the highest saturation magnetization (Ms) was measured for the smallest but well-crystalline NPs, while the larger NPs exhibiting worse crystallinity revealed a lower Ms. Our results clearly demonstrate that the NP crystallinity level that is mirrored in the internal spin order drives the specific magnetic response of the single-domain NPs.

Understanding particle size and distance driven competition of interparticle interactions and effective single-particle anisotropy.

Magnetic response of single-domain nanoparticles (NPs) in concentrated systems is strongly affected by mutual interparticle interactions. However, particle proximity significantly influences single-particle effective anisotropy. To solve which of these two phenomena plays a dominant role in the magnetic response of real NP systems, systematic study on samples with well-defined parameters is required. In our work, we prepared a series of nanocomposites constituted of highly-crystalline and well-isolated CoFe2O4 NPs embedded in an amorphous SiO2 matrix using a single-molecule precursor method. This preparation method enabled us to reach a wide interval of particle size and concentration. We observed that the characteristic parameters of the single-domain state (coercivity, blocking temperature) and dipole-dipole interaction energy ([Formula: see text]) scaled with each other and increased with increasing [Formula: see text], where d XRD was the NP diameter and r was the interparticle distance. Our results are in excellent agreement with Monte-Carlo simulations of the particle growth. Moreover, we demonstrated that the contribution of [Formula: see text] acting as an additional energetic barrier to the superspin reversal or as an average static field did not sufficiently explain how the concentrated NP systems responded to an external magnetic field. Alternations in the blocking temperature and coercivity of our NP systems accounted for reformed relaxations of the NP superspins and modified effective anisotropy energy of the interacting NPs. Therefore, the concept of modified NP effective anisotropy explains the magnetic response of our concentrated NP systems better than the concept of the energy barrier influenced by interparticle interactions.

A comparative study of meiotic recombination in cattle (Bos taurus) and three wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus).

The karyotypic evolution in the family Bovidae is based on centric fusions of ancestral acrocentric chromosomes. Here, the frequency and distribution of meiotic recombination was analyzed in pachytene spermatocytes from Bos taurus (2n = 60) and 3 wildebeest species (Connochaetes gnou, C. taurinus taurinus and C. t. albojubatus) (2n = 58) using immunofluorescence and fluorescence in situ hybridization. Significant differences in mean numbers of recombination events per cell were observed between B. taurus and members of the genus Connochaetes (47.2 vs. 43.7, p < 0.001). The number of MLH1 foci was significantly correlated with the length of the autosomal synaptonemal complexes. The average interfocus distance was influenced by interference. The male recombination maps of bovine chromosomes 2 and 25 and of their fused homologues in wildebeests were constructed. A significant reduction of recombination in the fused chromosome BTA25 was observed in wildebeests (p = 0.005). This was probably caused by interference acting across the centromere, which was significantly stronger than the intra-arm interference. This comparative meiotic study showed significant differences among the species from the family Bovidae with the same fundamental number of autosomal arms (FNa = 29) which differ by a single centric fusion.

Subchromosomal karyotype evolution in Equidae.

Equidae is a small family which comprises horses, African and Asiatic asses, and zebras. Despite equids having diverged quite recently, their karyotypes underwent rapid evolution which resulted in extensive differences among chromosome complements in respective species. Comparative mapping using whole-chromosome painting probes delineated genome-wide chromosome homologies among extant equids, enabling us to trace chromosome rearrangements that occurred during evolution. In the present study, we performed subchromosomal comparative mapping among seven Equidae species, representing the whole family. Region-specific painting and bacterial artificial chromosome probes were used to determine the orientation of evolutionarily conserved segments with respect to centromere positions. This allowed assessment of the configuration of all fusions occurring during the evolution of Equidae, as well as revealing discrepancies in centromere location caused by centromere repositioning or inversions. Our results indicate that the prevailing type of fusion in Equidae is centric fusion. Tandem fusions of the type telomere-telomere occur almost exclusively in the karyotype of Hartmann's zebra and are characteristic of this species' evolution. We revealed inversions in segments homologous to horse chromosomes 3p/10p and 13 in zebras and confirmed inversions in segments 4/31 in African ass, 7 in horse and 8p/20 in zebras. Furthermore, our mapping results suggested that centromere repositioning events occurred in segments homologous to horse chromosomes 7, 8q, 10p and 19 in the African ass and an element homologous to horse chromosome 16 in Asiatic asses. Centromere repositioning in chromosome 1 resulted in three different chromosome types occurring in extant species. Heterozygosity of the centromere position of this chromosome was observed in the kiang. Other subtle changes in centromere position were described in several evolutionary conserved chromosomal segments, suggesting that tiny centromere repositioning or pericentric inversions are quite frequent in zebras and asses.

Comparative molecular cytogenetics in Cetartiodactyla.

Cetartiodactyla comprises Artiodactyla (even-toed ungulates) and Cetacea (whales, dolphins and porpoises). Artiodactyla is a large taxon represented by about 200 living species ranked in 10 families. Cetacea are classified into 13 families with almost 80 species. Many publications concerning karyotypic relationships in Cetartiodactyla have been published in previous decades. Formerly, the karyotypes of closely related species were compared by chromosome banding. Introduction of molecular cytogenetic methods facilitated comparative mapping between species with highly rearranged karyotypes and distantly related species. Such information is a prerequisite for the understanding of karyotypic phylogeny and the reconstruction of the karyotypes of common ancestors. This study summarizes the data on chromosome evolution in Cetartiodactyla, mainly derived from molecular cytogenetic studies. Traditionally, phylogenetic relationships of most groups have been estimated using morphological data. However, the results of some molecular studies of mammalian phylogeny are discordant with traditional conceptions of phylogeny. Cetartiodactyls provide several examples of incongruence between traditional morphological and molecular data. Such cases of conflict include the relationships of the major clades of artiodactyls, the relationships among the extant families of the suborder Ruminantia or the phylogeny of the family Bovidae. The most unexpected aspect of the molecular phylogeny was the recognition that Cetacea is a deeply nested member of Artiodactyla. The largest living order of terrestrial hoofed mammals is the even-toed hoofed mammals, or Artiodactyla. The artiodactyls are composed of over 190 living species including pigs, peccaries, hippos, camels, llamas, deer, pronghorns, giraffes, sheep, goats, cattle and antelopes. Cetacea is an order of wholly aquatic mammals, which include whales, dolphins and porpoises. Cetartiodactyla has become the generally accepted name for the clade containing both of these orders.

Molecular insights into X;BTA5 chromosome rearrangements in the tribe Antilopini (Bovidae).

For a clade that includes Antilope, Gazella,Nanger and Eudorcas (Antilopinae), X;BTA5 translocation is a synapomorphy. Using a combination of fluorescence in situ hybridization (FISH) probes and polymerase chain reaction techniques, we provide (i) the first insight into the X;BTA5 architecture which differs in the species under study: Antilope cervicapra (genus Antilope), Gazella leptoceros (genus Gazella) and Nanger dama ruficollis (genus Nanger), (ii) determination of interstitial satellite DNA at the X;BTA5 junctions, and (iii) determination of repetitive sequences occupying constitutive heterochromatin of Xp arms in the studied species. The distribution of 2 repetitive DNA families in the centromeric regions of all chromosomes has been investigated by FISH with probes representing satellite I and satellite II DNA in all studied species. In this context, we discuss a markedly smaller centromere in the BTA5 (Y2) unfused chromosomes in males in the XY1Y2 determining system in comparison with other acrocentrics. An analysis of karyotypic data described in current published studies revealed a disparity with the data determined by FISH. In this report, we document chromosomal fusions in the 3 species mentioned resulting from FISH with painting probes prepared from cattle (Bos taurus). The number and chromosomal location of nucleolus organizer regions were determined by FISH. In the present study, we emphasize the importance of chromosomal rearrangement verification, particularly, if they are used for phylogenetic analysis.

Disturbances of nuclear maturation in BCB positive oocytes collected from peri-pubertal gilts.

The developmental competence (quality) of oocytes is affected by several factors linked to their intrinsic properties and also to growth and maturation environment. Donor puberty and chromosomal complement are one of the main factors influencing oocyte quality. A high rate of porcine oocytes matured in vitro is chromosomally imbalanced. Moreover, there is no published data on chromosomal aberrations in oocytes selected by the brilliant cresyl blue (BCB) test. Therefore, the aim of this study was to analyze whether BCB positive (BCB+) oocytes derived from ovaries of peripubertal gilts (prepubertal NCL and cyclic CL) differ with respect to the incidence of numerical chromosome aberrations. COCs collected from NCL and CL ovaries were selected by the BCB test. Only BCB+ oocytes were matured in vitro and subjected to FISH analysis using molecular probes for chromosome pairs 1 and 10. The rate of BCB+ oocytes was similar for both groups of ovaries (NCL 80%, CL 92%). Altogether 554 oocytes were fixed and 471 oocytes at the MII stage were analyzed cytogenetically. Diploid (2MII) and aneuploid oocytes were detected. The contribution of MII oocytes was similar for NCL (85%) and CL (90%) group. Chromosomally aberrant BCB+ oocytes accounted for 18.0% and ranged from 13.7% for CL and 22.2% for NCL ovaries. Diploidy was a predominant anomaly observed (11.2%) with a significantly higher frequency in BCB+ oocytes of pre-pubertal (16.7%) than cyclic gilts (5.6%, P < 0.05). Aneuploid oocytes occurred with similar rate in NCL (6.7%) and CL (8.5%) females. The majority of aneuploid spreads (72.2%; P < 0.01) concerned the chromosome pair 10. The overall rate of disomy (56%) and nullisomy (44.4%) was similar. We have shown that donor puberty affects the incidence of chromosomal abnormalities in porcine oocytes matured in vitro. Significantly more diploid oocytes was derived from prepubertal ovaries, whereas the frequency of aneuploidy was similar in NCL and CL gilts.

Cytotypes of Kirk's dik-dik (Madoqua kirkii,Bovidae) show multiple tandem fusions.

Madoqua kirkii, a miniature African antelope, is noted for extensive chromosomal variation that has been categorized in four distinct cytotypes (A-D). In this investigation, we analyzed the A cytotype (2n = 46, FN = 48) using a suite of molecular cytogenetic approaches that entailed (i) whole chromosome and subchromosomal painting by fluorescence in situ hybridization (FISH), (ii) the study of Madoqua centromeric-specific DNA derived from pooled DNA obtained from the centromeric regions of the acrocentric chromosomes, and (iii) DNA from the telomere:centromere junctions of tandemly fused chromosomes. DNA from these sources was used to probe for the persistence of interstitial satellite DNA and residual centromeric sequences in the tandem and centric fusion junctions by PCR and FISH. The analyses show centromeric sequences at two of the six tandem fusion junctions. These data, and those of hybrid specimens (A × B cytotypes) in conjunction with published information permitted an interpretation of the probable sequence of chromosomal rearrangements among the M. kirkii cytotypes. We discuss the findings in the context of chromosomal evolution in these antelopes, and the implications that these hold for ex-situ breeding programs of the species.

Different fusion configurations of evolutionarily conserved segments in karyotypes of Potamochoerus porcus and Phacochoerus africanus.

The karyotype of the red river hog Potamochoerus porcus (2n = 34) differs from that of the domestic pig by the presence of 2 fusion chromosomes homologous to pig chromosomes 13/16 and 15/17. Moreover, chromosomes corresponding to pig chromosomes 13/16 and 1 are both acrocentric. Hybridization with region-specific painting probes confirmed tandem fusion of pig chromosomes 13 and 16, and a pericentric inversion of the pig chromosome 1p equivalent in P. porcus. The chromosome complement of the wart hog Phacochoerus africanus (2n = 34) differs from the pig karyotype in 2 centric fusions, 13/16 and 15/17. Karyotypic relationships among different Suidae species are discussed in the article. Besides fusions 13/16 and 15/17, which are common to several suids, another fusion of pig chromosomes 14 and 18 is suggested to exist in the karyotype of Sus cebifrons.

Fertile bull sperm aneuploidy and chromatin integrity in relationship to fertility.

Aneuploidy is associated with spontaneous abortions, perinatal mortality, mental retardation and with embryonic and foetal mortality. Most of these abnormalities originate as a result of meiosis errors during gametogenesis. The main purpose of the study was to analyse frequency of aneuploidies of sex chromosomes and chromosome 6 by three-colour fluorescence in situ hybridization (FISH) in 47 young bulls, candidates for artificial insemination programme with cryopreserved semen and to investigate the influence of aneuploidies and disturbed sperm chromatin integrity on non-return rates, the frequencies of abortions, perinatal mortality and stillbirths. The average frequencies of spermatozoa with disomy for chromosomes X, Y, XY and 6 were 0.032, 0.005, 0.003 and 0.039% respectively. The incidence of XX66, YY66 and XY66 diploidy was 0.017, 0.006 and 0.015% respectively. Frequencies of meiotic II errors were significantly higher than meiotic I errors (p < 0.01). More X bearing spermatozoa than Y bearing spermatozoa were detected (5151 vs. 5022; p < 0.01). Sperm chromatin damage expressed by DNA fragmentation index (DFI) was 5.3 +/- 3.7 and percentage of cells with defective chromatin condensation (HDS) was 1.4 +/- 0.8. No correlation was found between sperm aneuploidy and basic sperm analysis. The relationship was found between non-return rate and total aneuploidy (r = -0.310; p = 0.036). Significant correlation was found between sex disomy, total aneuploidy (disomy of chromosomes 6, X, Y and XY spermatozoa and diploidy) and stillbirths (r = 0.390; p = 0.013; and r = 0.331; p = 0.037). Chromosome 6 disomy correlated with perinatal mortality (r = 0.317; p = 0.047). HDS correlated significantly with total aneuploidy (r = 0.449; p = 0.002). Our study indicated that aneuploidy frequencies in young fertile bull spermatozoa are relatively low. Nevertheless, there exists a variability in aneuploidy frequencies amongst bulls, which appears to be able to have an influence on the fertility of these animals.

Fluorescence in situ hybridization applied to domestic animal cytogenetics.

The aim of this article is not to present an exhaustive review of molecular cytogenetics applications in domestic animal species, but more to illustrate the considerable contribution of these approaches in diagnostics and research in economically important species. A short presentation of the main applications of molecular cytogenetics in humans points out the domains in which it has become an essential tool and underlines the specificities attached to this species in comparison to farm animals. This article is devoted to outlining the current resources available in domestic species and to some examples of fluorescence in situ hybridization applications in the cattle, pig, horse and avian species. From a clinical point of view, these examples illustrate the advantages of FISH for the study of chromosomal abnormalities (identification, characterization and estimation of their effects). Other applications of molecular cytogenetics are also illustrated, particularly ZOO-FISH, an approach which allows the determination of chromosome homologies between species. Finally, a specific emphasis was placed on the usefulness of molecular cytogenetics for the analysis of species such as poultry, which harbour a complex karyotype.

Investigation of chromosome aneuploidies in early porcine embryos using comparative genomic hybridization.

Although numerical chromosome errors are known to be prevalent in early human embryos and are likely to be a considerable factor influencing the mortality of early embryos and implantation failure, in domestic animals data about the frequency and nature of errors is limited. The objectives of this study were to investigate the whole chromosome set of in vivo obtained early pig embryos, applying methods of whole genome amplification and comparative genomic hybridization (CGH) and to contribute to the comprehensive understanding of the topic. The embryos were collected from gilts 72 h after insemination. Further, they were lysed and underwent whole genome amplification by multiple displacement amplification. In a subsequent CGH, amplified DNA was compared to control DNA using different fluorescent labeling and hybridization to male pig mitoses. 11 (14.3%) of the 77 pig embryos examined were observed to be aneuploid. We found chromosome errors comprising loss/gain of one or a few chromosomes (10.4%) but also extensive chromosome imbalances (3.9%). Chromosomes 8, 11, 12, 13, 17, and X were most frequently involved in aneuploidies, when compared to chromosomes 2, 9, and 18, which were rarely involved in chromosome errors.

Sex chromosome evolution in cotton stainers of the genus Dysdercus (Heteroptera: Pyrrhocoridae).

The neo-X and neo-Y sex chromosomes of Dysdercus albofasciatus represent a unique model for the study of early stages of sex chromosome evolution since they retained the ability to pair and recombine, in contrast to sex chromosomes in most Heteroptera. Here we examined structure, molecular differentiation, and meiotic behaviour of the D. albofasciatus neo-sex chromosomes. Two related species with the ancestral X0 system, D. chaquensis and D. ruficollis, were used for a comparison. In D. albofasciatus, 2 nucleolar organizer regions (NORs) were identified on the neo-X chromosome using fluorescence in situ hybridization (FISH) with an rDNA probe, whereas a single NOR was found on an autosomal pair in the other 2 species. Genomic in situ hybridization (GISH) differentiated a part of the original X in the neo-X chromosome but not the neo-Y chromosome. The same segment of the neo-X chromosome was identified by Zoo-FISH with a chromosome painting probe derived from the X chromosome of D. ruficollis, indicating that this part is conserved between the species. Immunostaining against the cohesin subunit SMC3 revealed that only terminal regions of the D. albofasciatus neo-Xneo-Y bivalent pair and form a synaptonemal complex, which is in keeping with the occurrence of terminal chiasmata, whereas the interstitial region forms a large loop indicating the absence of homology. These results support the hypothesis that the neo-X chromosome evolved by insertion of the original X chromosome into 1 NOR-bearing autosome in an ancestor carrying the X0 system. As a consequence, the homologue of this NOR-autosome became the neo-Y chromosome. A subsequent inversion followed by transposition of the NOR located on the neo-Y onto the neo-X chromosome resulted in the present neo-sex chromosome system in D. albofasciatus.

Single nucleotide polymorphisms of interleukin-1 beta related genes and their associations with infection in the horse.

In previous work, we found significant associations of horse chromosome 15 (ECA15) microsatellite markers HMSO1 and HTG06 with two horse infections, Rhodococcus equi and Lawsonia intracellularis, respectively. Interleukin-1 beta subunit and interleukin-1 receptor antagonist encoding genes (IL1B and IL1RN) could be considered as candidate genes underlying the associations reported. Therefore, we identified single nucleotide polymorphisms (SNPs) within three interleukin-1 beta functionally related genes: IL1B, IL1RN and Casp1 (interleukin-1 beta converting enzyme/caspasel encoding gene). Using appropriate restriction fragment length polymorphism (PCR-RFLP) and/or single strand conformation polymorphism (PCR-SSCP) markers, their associations with the two infections by genotyping foals from the original study were tested. In addition, the physical localization of one of the two closely located genes, IL1RN, was re-assessed by fluorescence in-situ hybridization (FISH). A statistically significant association between an intronic SNP of the Casp1 gene with R. equi infection was found. The IL1RN gene was localized to 15q13-q14 in agreement with its originally reported physical position.

Detection of translocation rob(1;29) in bull sperm using a specific DNA probe.

The Robertsonian translocation rob(1;29), connected with reduced fertility, is widespread in different cattle breeds all over the world. After laser microdissection, DOP-PCR, cloning and sequencing, a highly sensitive translocation-specific DNA probe, suitable for detection of rob(1;29) in cattle metaphase and interphase cells, including spermatozoa was designed. Sperm samples of five heterozygous translocation carriers were analyzed using this probe and a control probe for chromosome 6. One thousand decondensed spermatozoa from each bull were scored. Signals of the translocation-specific probe were detected in 48.8, 50.9, 50.1, 51.8, and 54.8% of spermatozoa, respectively. In contrast, semen samples from five chromosomally normal bulls showed only signals of the control probe for chromosome 6. Semen from a chimeric (XX/XY) bull, showing 57.5% of 59,XX,rob(1;29) and 42.5% of 60,XY cells in cultured peripheral lymphocytes, was also examined using this probe. No sperm head with signal of the translocation-specific probe was observed among 1,000 spermatozoa analyzed in this bull, demonstrating that female cells do not pass through the process of spermatogenesis.

Karyotypic relationships among Equus grevyi, Equus burchelli and domestic horse defined using horse chromosome arm-specific probes.

Using laser microdissection we prepared a set of horse chromosome arm-specific probes. Most of the probes were generated from horse chromosomes, some of them were derived from Equus zebra hartmannae. The set of probes were hybridized onto E. grevyi chromosomes in order to establish a genome-wide chromosomal correspondence between this zebra and horse. The use of arm-specific probes provided us with more information on the mutual arrangement of the genomes than we could obtain by means of whole-chromosome paints generated by flow sorting, even if we used reciprocal painting with probe sets from both species. By comparison of our results and results of comparative mapping in E. burchelli, we also established the chromosomal correspondence between E. grevyi and E. burchelli, providing evidence for a very close karyotypic relationship between these two zebra species. Establishment of the comparative map for E. grevyi contributes to the knowledge of the karyotypic phylogeny in the Equidae family.