D. Di Berardino

Genotoxicity and oxidative stress induced by pesticide exposure in bovine lymphocyte cultures in vitro.

The genotoxic activity of the pesticides gliphosate, vinclozolin and DPX-E9636 was studied in in vitro cultures of bovine lymphocytes, using chromosome aberration (CA) and sister chromatid exchange (SCE) frequencies as genetic end-points and a variation of glucose 6-phosphate dehydrogenase (G6PD) enzyme activity as a marker of changes in the normal cell redox state. Results indicated a statistically significant increase of structural aberrations, sister chromatid exchanges and G6PD activity, suggesting that the pesticides tested induce either oxidative stress or a mutagenic effect in this species. The evaluation of both mitotic index and cell viability, after pesticide exposure, demonstrates a high cytotoxic effect which is always associated with the observed genotoxic effect.

Chromosomal abnormalities in Day-6, in vitro-produced pig embryos

A cytogenetic study was undertaken to quantify, by chromosomal karyotyping, the incidence and type of chromosomal abnormalities present in Day-6 in vitro-produced (IVP) porcine embryos. Morphologically normal Day-6 blastocysts (n=318) were fixed and grouped into six classes according to the number of total cells (from < or =20 to 61-70). Of 248 embryos suitable for analysis, 97 (39.1%) displayed chromosomal abnormalities. The abnormalities included haploidy (9.3%), polyploidy (71.1%) and mixoploidy (19.6%). Within polyploid embryos, triploidy and tetraploidy showed the highest incidence (56.5 and 27.5%, respectively); among mixoploid embryos, diploid-triploid embryos (2n/3n) were prevalent (36.8%). Overall, the mean cell number was 34.3 +/- 12.1 and the mitotic index was 8.6 +/- 6.1. Chromosomally abnormal embryos had fewer (P<0.01) total cells compared to normal (2n) embryos (31.8 +/- 1.3 versus 35.9 +/- 1.0). In addition, the incidence of polyploidy decreased as the number of cells increased, while that of mixoploidy did not differ. These data indicate that polyploidy affects a large percentage of IVP porcine embryos capable of developing to blastocysts and the incidence of chromosomal abnormalities is much higher than that reported previously in in vivo embryos in this species. Given the ability of morphologically normal embryos with an abnormal chromosome complement to undergo preimplantation development in vitro, and the inability to identify blastocysts with abnormal karyotype without cytogenetic analysis, careful consideration should be given to factors affecting ploidy of IVP embryos, especially the incidence of polyspermic fertilization, when evaluating criteria of a porcine in vitro embryo production scheme.

Chromosomal aberrations induced by ELF electric fields

Bovine lymphocytes in McCoy culture medium and autologous plasma were exposed to 50 Hz 2.4 µA/cm2 current density. Chromosomal aberrations (breaks, aneuploidy, ployploidy, deletions, fragments) were significantly increased in exposed cultures. The number of sister chromatid exchanges was unchanged.

Chromosome evolution in domestic bovids as revealed by chromosome banding and FISH-mapping techniques.

The present review summarizes the basic cytogenetic information available pertaining to the most important Bovidae species, namely cattle, buffalo, sheep and goat, with the aim of tracing their evolutionary relationships and to provide – for the first time – the hypothetical ancestral karyotype of the Bovinae-Caprinae subfamilies, also in relation to the other nondomestic species which are included in this important taxonomic family. Evolution of the Bovinae-Caprinae autosomes and gonosomes is discussed on the basis of the most recent advances in chromosome banding, linkage studies, FISH-mapping and molecular information.

An extended river buffalo (Bubalus bubalis, 2n=50) cytogenetic map: assignment of 68 autosomal loci by FISH-mapping and R-banding and comparison with human chromosomes

We report an extended river buffalo (Bubalus bubalis, 2n = 50; BBU) cytogenetic map including 388 loci, of which 68 have been FISH-mapped on autosomes in the present study. Ovine and caprine BAC clones containing both type I loci (known genes) and type II loci (simple sequence repeats (SRs), microsatellite marker, sequence-tagged sites (STSs)), previously assigned to sheep chromosomes, have been localized on R-banded river buffalo chromosomes (BBU), which expands the cytogenetic map of this important domestic species and increases our knowledge of the physical organization of its genome. The loci mapped in the present study correspond to loci already localized on homoeologous cattle (and sheep) chromosomes and chromosome bands, further confirming the high degree of chromosome homoeologies among bovids. The comparison of the integrated cytogenetic maps of BBU2p/BBU10 and BBU5p/BBU16 with those of human chromosomes (HSA) 6 and 11, respectively, identified, at least, nine conserved chromosome segments in each case and complex rearrangements differentiating river buffalo (and cattle) and human chromosomes.

Localization of BrdU-Induced Break Sites in Bovine Chromosomes

SUMMARYA map of BrdU-induced breaks in chromosomes of cattle has been achieved by using late BrdU incorporation for producing RBA bands in lymphocytes of a female calf affected by chromosome instability. This procedure gave a conspicuous rate of chromosome breaks which were carefully located on a diagrammatic representation of the RBA banded bovine karyotype arranged according to the Reading nomenclature. The sites of BrdU-induced breaks were found to be located at the junctions between R-positive (G-Q negative) and R-negative (G-Q positive) bands and, sometimes, between the centromere and the first subcentromeric band. The distribution of breaks among the chromosomes was found to be proportional to chromosome length. Highly sensitive BardU-break sites have been found in chromosomes 2, 12 and on the short arm of the early replicating X chromosome.

Karyotype, centric fusion polymorphism and chromosomal aberrations in captive-born mountain reedbuck (Redunca fulvorufula)

Chromosomes of fourteen captive-born mountain reedbucks (Redunca fulvorufula) have been investigated. The diploid chromosome number was 2n = 56 (FN = 60). The mountain reedbuck karyotype consists of 26 acrocentric and two biarmed chromosome pairs resulting from two centric fusions involving chromosomes 2 and 25, and 6 and 10, respectively. In some animals, 57 chromosomes were detected. Variation in the diploid number was found to be due to polymorphism for the centric fusion 6;10. Both X and Y chromosomes are large and acrocentric. The entire Y chromosome and the proximal part of the X chromosome consist of heterochromatin. The chromosomes X, 9 and 14 appeared to be of caprine type. Chromosome aberrations have been detected in two of the 14 animals investigated. A de novo formed Robertsonian translocation rob(6;13) was found in one female heterozygous for the fusion 6;10. CBG-banding revealed one block of centromeric heterochromatin in the de novo formed translocation rob(6;13) and also in the evolutionarily fixed centric fusions 6;10 and 2;25. One examined male homozygous for fusion 6;10, had a mosaic 56,XY/57,XYY karyotype, with 11% of analyzed cells containing two Y chromosomes. The findings were confirmed by cross-species fluorescence in situ hybridization (FISH) with bovine (Bos taurus L.) chromosome painting probes. The study demonstrates the relevance of cytogenetic screening in captive animals from zoological gardens.

The high-resolution RBA-banding pattern of bovine chromosomes

The high-resolution RBA-banding pattern of bovine chromosomes (Bos taurus L.), together with a diagrammatic representation, are presented. The haploid set of prometaphase chromosomes shows 521 bands, 183 of which are positive, 257 negative, and 81 variable. Compared to early metaphase chromosomes, the prometaphase chromosomes show 50% more bands, resulting in a more accurate description of the banding pattern of individual chromosomes of this species.

Identification of Nucleolus Organizer Chromosomes in Cattle (Bos Taurus L.) by Sequential Silver Staining + Rba Banding

SUMMARYA sequential «silver staining + RBA banding» procedure was applied to cytological preparations from six individuals of the Friesian breed of Bos taurus L. (2n = 60) for the identification of nucleolus organizer chromosomes. Chromosome identification followed the Reading system. Of 10 sequentially stained metaphase plates for each animal investigated, Ag-NORs were located on the telomeres of five pairs of autosomes identified as nos. 2, 3, 6, 11 and 27, indicating that two of the five NOR chromosomes in cattle have previously been misidentified.

Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes

The present study provides specific cytogenetic information on prometaphase chromosomes of the alpaca (Lama pacos, fam. Camelidae, 2n = 74) that forms a basis for future work on karyotype standardization and gene mapping of the species, as well as for comparative studies and future genetic improvement programs within the family Camelidae. Based on the centromeric index (CI) measurements, alpaca chromosomes have been classified into four groups: group A, subtelocentrics, from pair 1 to 10; group B, telocentrics, from pair 11 to 20; group C, submetacentrics, from pair 21 to 29; group D, metacentrics, from pair 30 to 36 plus sex chromosomes. For each chromosome pair, the following data are provided: relative chromosome length, centromeric index, conventional Giemsa staining, sequential QFQ/C-banding, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization. The overall number of RBG-bands revealed was 391. Nucleolus organizer-bearing chromosomes were identified as pairs 6, 28, 31, 32, 33 and 34. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X and Y painting probes was also carried out to validate X-Y chromosome identification of alpaca and to confirm close homologies between the sex chromosomes of these two species.