Nucleolar organizer regions (NORs) distribution and behavior in spermatozoa and meiotic cells of the horse (Equus caballus).

Nucleolar organizing regions (NORs) containing rDNA gene clusters have been assigned to the equine autosomes ECA1, ECA28, and ECA31. Active NORs (Ag-NORs) are associated with argyrophilic proteins, which allow them to be readily identified using silver staining techniques. Fluorescence in situ hybridization (FISH) for rDNA can also be used to visualize all NOR clusters in the nucleus, regardless of whether they are active or inactive. The present study analyzed the distribution and behavior of equine Ag-NOR and NOR clusters in horse spermatozoa and during male meiosis by FISH and silver staining. The NOR foci were observed to be variable in number, size, and shape, but were usually located centrally and appeared as one or two nucleolus-like structures in the spermatozoa head. Three distinctive FISH signals identified the NOR-bearing chromosome pairs during the synaptic cell stage of meiosis I. At diakinesis/metaphase I, as well as different stages of meiosis II, FISH signals clearly depicted the NOR-bearing sister chromatids. The synaptonemal complexes of primary spermatocytes consistently showed three rDNA foci following FISH, but variably demonstrated two or three Ag-NOR bodies following silver staining. We propose rDNA loss and gain during unequal crossing-over events could be both a direct and indirect cause of variation in equine NOR foci. Additionally, our cytogenetic analysis did not confirm the presence of a fourth pair of NORs-bearing chromosomes in the horse, which is contrary to previously mitotic published data.

Determination of the correlation between stallion's age and number of sex chromosome aberrations in spermatozoa.

The aim of this study was a cytogenetic analysis of stallions semen to find sex chromosome aberrations and to determine if there was an association between stallion's age and aberration frequency for the sex chromosomes. Sperm samples were collected from 22 stallions of various age from 3 to 23 years. Multicolour FISH was performed on each sample, using probes for the sex chromosomes and EGFR gene, localized on 4p12 in domestic horse. A total of 26199 sperm cells were analysed (from 1 070 to 1 532 per animal). Among the analysed cells, there were 50.318% with X chromosome, 48.543% with Y chromosome and 1.139% with aberrant chromosomes. The frequency of aberrations was: sex chromosomes nullisomy (0.466%), XY aneuploidy (0.454%), XX disomy (0.146%), YY disomy (0.041%), diploidy (0.024%) and trisomy XXY (0.008%). Additionally there was a correlation between the age of an animal and the frequency of sex chromosome aberration and a significant positive correlation between age and disomy of XY, XX, YY, trisomy of XXY, autosomal disomy was seen. A Correlation between the age of a stallion and the level of nullisomy was negative. The present study demonstrated that FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and might be very helpful for a breeder during a selection for the best stallion.

Usefulness of a supplementary set of microsatellite DNA markers for parentage testing in cattle.

The aim of the present study was to determine a supplementary set of microsatellite DNA markers to be used in addition to the standard panel of 12 microsatellite markers recommended for parentage testing in cattle. A method has been developed for analysis of 10 loci in a single multiplex PCR reaction and using automated DNA sizing technology in a sequencer, which will enable the additional set of markers to be used for routine pedigree verification. Preliminary analysis showed that the polymorphism of 10 selected markers (PIC and H > 0.5 for 9 loci) and PEc of 99.77% suggest that the microsatellite sequences selected could be useful in cases of disputable parentage where the standard set may prove insufficient, e.g. in closely related animals or those from herds subjected to rigorous selection.

Detection of sex chromosome aneuploidy in equine spermatozoa using fluorescence in situ hybridization.

The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X- and Y-bearing cells was different from the expected 1:1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1:1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X-Y-spermatozoa.

Comparative cytogenetic and molecular studies of DM domain genes in pig and cattle.

Despite great differences in the biology of sex determination, the DM domain genes are thought to regulate sexual development in both invertebrates and vertebrates. Although they seem to represent the first known example of the conservation of metazoan sexual pathways, the structure-function relationships of this gene family have not yet been clearly determined. Our previous and current studies on the DMRT (Doublesex and mab-3 related transcription factor) gene cluster, DMRT1, DMRT2, DMRT3, in pig and cattle, have revealed conserved linkage to bovine (BTA8q17) and swine (SSC1q21) autosomes. However, the DMRT cluster seems to harbour the genes in different orders in both cattle (Entrez database) and pig (IMpRH map), compared to other vertebrates investigated so far. Moreover, we found that expression of DMRT1 (considered as the testicular regulator so far) is not restricted to gonads in adult animals. Beside ovary and testis, DMRT1 expression was detectable in swine kidney and bovine liver, heart, skeletal muscle, kidney, lung, spleen and showed no differences between the sexes. Swine and bovine DMRT3 expression was strongly restricted to the adult testis, while DMRT2 homologues have a wider expression pattern, as occurs in other mammals. The significance of this expression pattern of DMRT genes in adult pig and cattle remains unclear. It might shed new light on mammalian DM domain genes evolution and livestock intersexuality.

Interleukin 4 receptor alpha (IL4R) and calcium-activated chloride channel 1 (CLCA1) genes map to donkey chromosome.

The results obtained in the present study enabled the physical map of the donkey genome to be extended with markers associated with recurrent airway obstruction (RAO), a major performance-limiting disease of Equidae. The equine BAC clone containing the IL4R and CLCA1 genes were localized to EAS 14q13 and EAS 6q15 respectlivy by fluorescent in situ hybridization. Identification of their locus confirmed the distribution of syntenic regions between the domestic horse and the domestic donkey within the chromosomes analysed.

Application of 19 microsatellite DNA markers for parentage control in Borzoi dogs.

The aim of this study was to analyse the polymorphism of 19 microsatellite markers and their usefulness for parentage verification in 28 Borzoi dogs in Poland. The assay involved the following 19 loci: AHTk211, CXX279, REN169018, INU055, REN54P11, INRA21, AHT137, REN169D01, AHTh260, AHTk253, INU005, INU030, FH2848, AHT121, FH2054, REN162C04, AHThl71, REN247M23 and amelogenin. These markers are included in the panel of loci recommended by the ISAG. Amplified PCR products were analysed by an automated sequencer ABI PRISM 3130xl Genetic Analyser. The number of alleles per locus varied from 3 (INU005) to 8 (REN162 and AHT171). Except the INU005 locus, which showed the lowest variation (PIC=0.396, H=0.438), the calculated PIC values exceeded 0.6 and H values ranged from 0.677 (INU055) to as much as 0.84 (REN162). Based on PE(C) it was found that incorrect pedigree in this breed can be excluded with 99.9998% accuracy using DNA analysis.

Identification of carriers of the mutation causing coagulation factor XI deficiency in Polish Holstein-Friesian cattle.

Factor XI (FXI) deficiency is a hereditary coagulation disorder observed in various mammalian species. The molecular basis of coagulopathy has been recognized in Holstein cattle as a 76-bp insertion in the coding region of the FXI gene. Because the disorder seems to have an impact on reproductive traits and udder health in cattle, we tested 103 randomly selected cows, 28 cows with repeat breeding, and 9 cows with recurrent mastitis for the presence of an abnormal FXI allele. Three related cows were diagnosed as carriers.

Cytogenetic screening of livestock populations in Europe: an overview.

Clinical animal cytogenetics development began in the 1960's, almost at the same time as human cytogenetics. However, the development of the two disciplines has been very different during the last four decades. Clinical animal cytogenetics reached its 'Golden Age' at the end of the 1980's. The majority of the laboratories, as well as the main screening programs in farm animal species, presented in this review, were implemented during that period, under the guidance of some historical leaders, the first of whom was Ingemar Gustavsson. Over the past 40 years, hundreds of scientific publications reporting original chromosomal abnormalities generally associated with clinical disorders (mainly fertility impairment) have been published. Since the 1980's, the number of scientists involved in clinical animal cytogenetics has drastically decreased for different reasons and the activities in that field are now concentrated in only a few laboratories (10 to 15, mainly in Europe), some of which have become highly specialized. Currently between 8,000 and 10,000 chromosomal analyses are carried out each year worldwide, mainly in cattle, pigs, and horses. About half of these analyses are performed in one French laboratory. Accurate estimates of the prevalence of chromosomal abnormalities in some populations are now available. For instance, one phenotypically normal pig in 200 controlled in France carries a structural chromosomal rearrangement. The frequency of the widespread 1;29 Robertsonian translocation in cattle has greatly decreased in most countries, but remains rather high in certain breeds (up to 20-25% in large beef cattle populations, even higher in some local breeds). The continuation, and in some instances the development of the chromosomal screening programs in farm animal populations allowed the implementation of new and original scientific projects, aimed at exploring some basic questions in the fields of chromosome and/or cell biology, thanks to easier access to interesting biological materials (germ cells, gametes, embryos ...).

A case of an intersex horse with 63,X/64,XX/65,XX,del(Y)(q?) karyotype.

Cytogenetic and molecular genetic studies of an intersex horse have been carried out. The investigated animal had overall male body conformation; however, its external genitalia consisted of incompletely developed vulva and penis. The X and Y chromosome painting probes detected three cell lines in the examined horse: 63,X, 64,XX and 65,XX with a fragment of a Y chromosome (del Y). The DNA analysis with the PCR and PCR/RFLP methods showed absence of SRY,AMELY and ZFY genes as well as of six Y microsatellite markers (YM2, YP9, YJ10, YE1, YH12, and YA16). These results suggest that the Y chromosome fragment detected in the investigated animal was the result of a deletion of a euchromatic fragment comprising the above-mentioned markers.

The mechanisms determining the nucleolar-organizing regions inactivation of domestic horse chromosomes.

Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with HpaII, silver staining (AgNOR) and C-banding technique (CBG). The obtained results suggest that variation in the number and size of silver deposits is related to the number of rDNA copies, DNA methylation and the localization of ribosomal DNA loci in telomeric regions. Moreover, we have found that chromosome pairs 28 and 31 are characterized by higher variation in the NORs number.

Fluorescent in situ hybridization mapping of the epidermal growth factor receptor gene in donkey.

The physical localization of the epidermal growth factor receptor (EGFR) gene was performed on donkey chromosomes. Bacterial artificial chromosome DNA containing the equine EGFR gene was used to map this gene by fluorescent in situ hybridization on donkey metaphase chromosomes. The gene was mapped on donkey 1q21.1 region.

Karyotype evaluation among young horse populations in Poland.

Five hundred young horses of the following breeds: Thoroughbred, Silesian, Malopolska, Wielkopolska, Polish Konik, Hutsul, Shetland Pony, Half-bred Anglo-Arabian, Noble Half-bred, Fjord and crosses were cytogenetically investigated. Chromosome preparations obtained after lymphocyte culture were analysed using conventional Giemsa staining and CBG-banding methods. In the case of abnormalities GTG-banding as well as FISH technique were applied. In ten mares different karyotypic abnormalities were diagnosed. One mare showed chromosome chimerism (64,XX/64,XY), eight had sex chromosomal aneuploidy (one in pure line 63,X and seven in mosaic form 63,X/64,XX) and one presented autosomal aneuploidy with mosaicism (64,XX/65,XX,+31). The influence of sex chromosome abnormalities on fertility and the possible utilisation of karyotypic control in any selection programme are discussed.

Comparison between the G-banded karyotype of the aoudad (Ammotragus lervia) and sheep (Ovis aries).

Karyotypes of the aoudad and sheep were compared on the basis of G-banded chromosomes at the 450 band level. The common G-banded karyotype showed the homology of all aoudad chromosomes (2n=58) with sheep chromosomes (2n=54) or sheep chromosome arms. The results of cytogenetic investigations suggest that in this case karyotype evolution has led to reduction in chromosome number as a result of centric fusions. The formation of the first metacentric chromosome occurred in the aoudad. The homology of the G-banding pattern in sheep and aoudad suggests the conservation in linear arrangement of genetic material. Thus comparative cytogenetics can be a useful tool in gene mapping.

60,XY/60,XX chimerism in the germ cell line of mature bulls born in heterosexual twinning.

According to present knowledge there is a germ cell chimerism (XY/XX) in young bulls born in heterosexual twinning due to exchange of primordial germ cells in embryonic life. These germ cells were believed to have been eliminated in the young bull. Two-color fluorescence in situ hybridization (FISH) identification of the sex chromosomes by biotinylated and digoxygenin labeled probes have been used. The material consisted of three bulls born in heterosexual twinning. The results obtained indicated that even mature bulls (more than two years old) demonstrate spermatogonial chimerism. Several authors state that the bulls with blood cell chimerism, originating from dizygous twinning, are characterized by decreased fertility. Changes of the sex ratio of offspring due to proliferation of the female cells have also been proposed. The present observations should give a renewed interest in checking the possibility of survival and differentiation of germ cells from the female partner in the germ cell lines.