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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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The development of molecular techniques with fluorochromes has had an invaluable impact on discovering the nature of chromatin structure. Here, we show the application of a locus specific identifier probe (LSI) for precise and selective visualization of the horse IGF2 gene in the metaphase, interphase nuclei and sperm cells. Our study may be helpful for interpretation of results of interphase fluorescence in situ hybridization (I-FISH). We analyze and discuss the variation in the number and localization of FISH signals in somatic and sperm cells of horse.

Antioxidant status of seminal plasma from 23 stallions was evaluated. We found a negative correlation between total antioxidant capacity (ABTS(•+) decolorization assay) and thiol content of seminal plasma, and sperm DNA damage (8-oxoG immunostaining, TUNEL reaction, comet assay). Low seminal redox status was the strongest correlated with 8-oxoG level which may indicate that seminal total antioxidant capacity influences mainly the formation of single strand DNA breaks in sperm cells. Since inter-individual differences in seminal antioxidant status were reported, we postulated that the redox status of seminal plasma may be an additional important parameter, both with sperm quantitative and morphological analysis, for evaluation of equine semen quality., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Fluorescence in situ hybridization (FISH) is widely used in the study of chromosome structure and organization. Cytogenetic evaluation of chromosomes using FISH technique plays an increasingly important role in diagnosing karyotype changes in both somatic and reproductive cells. The aim of the study was to optimize the conditions of stallion sperm decondensation, which have a significant effect on the results of fluorescence in situ hybridization. Appropriate type and time of decondensation was chosen for the sperm of every stallion. It was found that decondensation performed using a preparation incubated in DTT solution for 1.5 minutes and in SDS solution for 10 seconds proved effective for stallions no. 1 and 2. An alternative decondensation method performed in an Eppendorf tube, with incubation in DTT solution for 1 minute and in SDS solution for 5 seconds proved effective for stallions no. 3 and 4. Decondensation using DTT and papain solution, a method successfully used for bull spermatozoa, proved inadequate for horse spermatozoa.

Contents 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. [ABSTRACT FROM AUTHOR]