Dual-color high-resolution fiber-FISH analysis on lethal white syndrome carriers in sheep.

Molecular defects occurring in the endothelin receptor type-B (EDNRB) gene are known to be associated with pigmentary anomalies and intestinal aganglionosis in humans, rodents and horses. We carried out a cytogenetic investigation in 2 ewes heterozygous for the deletion of the EDNRB gene and in 2 more females as control. The RBA-banding showed that all 4 ewes were karyologically normal. EDNRB gene-specific probes were produced by PCR and cloning. The application of the R-banding and propidium iodide-staining fluorescent in situ hybridization allowed mapping the gene to OAR 10q22 and confirmed the heterozygous status of the ewes investigated for the EDNRB gene deletion. For the fine estimation of the gene length in sheep and for the correct sizing of the chromosomal gap, a dual-color FISH was applied to high-resolution DNA fibers in combination with digital imaging microscopy. The comparison of the DNA fiber barcodes indicated a chromosomal deletion larger than the EDNRB gene itself. The length of the gene, not known for sheep until now, was estimated to be ∼21 kb, whereas the microchromosomal deletion was ∼100 kb. EDNRB is located in a chromosomal region previously shown to be a fragile site. The applied method allowed locating the potential breakpoints, thus permitting further interesting prospective investigations also in the field of the fragile sites in sheep.

Cytogenetic elaboration of a novel reciprocal translocation in sheep.

Reciprocal translocations represent one of the most common structural chromosomal rearrangements observed in both humans and domestic animals. In these translocations, the balanced forms are most frequent but may remain undetected because the carriers show a normal phenotype. For this reason, routine cytogenetic analysis of domestic animals should necessarily rely on banded karyotypes. In fact, during a screening analysis, carried out on phenotypically normal young sheep (Ovis aries, OAR, 2n = 54) from Laticauda-Comisana hybrids, a new structural rearrangement was detected. Two abnormal acrocentric chromosomes (the smallest and the largest one) were found in all metaphases of this carrier animal, suggesting the presence of a reciprocal translocation (rcp). CBA and RBA banding were performed in order to characterize the translocation, and FISH with chromosome-specific BAC probes and telomere probes was applied to confirm the cytogenetic data. The translocation was classified as rcp(4q;12q)(q13;q25).

Advanced comparative cytogenetic analysis of X chromosomes in river buffalo, cattle, sheep, and human.

Based on a recently generated comprehensive gene map for Ovis aries chromosome X (OARX) with an approximately even locus distribution, we assigned selected bacterial artificial chromosome (BAC) probes corresponding to these OARX loci to Bubalus bubalis (BBU) and Bos taurus (BTA) by comparative fluorescence in-situ hybridization (FISH) to improve cytogenetically the X chromosome maps in these species. Twenty-five added loci in BBUX and BTAX, respectively, contribute to a more detailed description of the cytogenetic organization of these chromosomes. Further seven loci were identified in OARX and two DNA probes were assigned to X and Y chromosomes in river buffalo, cattle, and sheep, respectively, and thus identified loci in the pseudoautosomal region. The additional assignments double the number of cytogenetic loci in BBUX and increase their number in BTAX and OARX. The larger quantity of cytogenetic anchors allows a more precise morphological comparison of bovid X chromosomes among each other and with the Homo sapiens (HSA) X chromosome. The anchor loci confirm and refine syntenic fragments in HSAX and identify several evolutionary breakpoints between the compared chromosomes. The cytogenetic assignments in BBUX, BTAX, and OARX represent useable anchors for the ongoing genome sequence assembly in Bovidae.

Extended cytogenetic maps of sheep chromosome 1 and their cattle and river buffalo homoeologues: comparison with the OAR1 RH map and human chromosomes 2, 3, 21 and 1q.

Cytogenetic maps are useful tools for several applications, such as the physical anchoring of linkage and RH maps or genome sequence contigs to specific chromosome regions or the analysis of chromosome rearrangements. Recently, a detailed RH map was reported in OAR1. In the present study, we selected 38 markers equally distributed in this RH map for identification of ovine genomic DNA clones within the ovine BAC library CHORI-243 using the virtual sheep genome browser and performed FISH mapping for both comparison of OAR1 and homoeologous chromosomes BBU1q-BBU6 and BTA1-BTA3 and considerably extending the cytogenetic maps of the involved species-specific chromosomes. Comparison of the resulting maps with human-identified homology with HSA2q, HSA3, HSA21 and HSA1q reveals complex chromosome rearrangements differentiating human and bovid chromosomes. In addition, we identified 2 new small human segments from HSA2q and HSA3q conserved in the telomeric regions of OAR1p and homoeologous chromosome regions of BTA3 and BBU6, and OAR1q, respectively. Evaluation of the present OAR1 cytogenetic map and the OAR1 RH map supports previous RH assignments with 2 main exceptions. The 2 loci BMS4011 and CL638002 occupy inverted positions in these 2 maps.

A new and unusual reciprocal translocation in cattle: rcp(11;25)(q11;q14-21).

A new and unusual reciprocal translocation was detected in a heifer of the Agerolese cattle breed during a routine cytogenetic screening carried out on 13 animals (2 males and 11 females) kept at the ConSDABI Conservation Center in Benevento (Southern Italy). The 13 animals investigated had a normal karyotype except for a 1-year-old female, which carried one autosome smaller than the smallest normal bovine autosomes. This small autosome showed very little C-banding in comparison to the other autosomes, while another medium-sized autosome showed 2 distinct and prominent C-bands. RBA-banding and karyotype analysis revealed that these 2 chromosomes were the result of a reciprocal translocation between chromosomes 11 and 25. FISH analysis with BAC142G06 mapping to the proximal (subcentromeric) region of both BTA25 and der11, BAC513H08 (ELN) mapping to BTA25q22dist and der25, and BAC533C11 mapping to the proximal region of BTA11 and der11 confirmed the localization of the breakpoints on band q11 (centromere) of chromosome 11 and q14-21 of chromosome 25. Ag-NOR and sequential RBA/Ag-NOR techniques detected the presence of NORs on both BTA11 and BTA25 and both der11 and der25. To our knowledge, this is the first report of a reciprocal translocation event in cattle with the breakpoint located in the centromeric region.

Short communication: Translational efficiency of casein transcripts in Mediterranean river buffalo.

Buffalo milk is characterized by the presence of all 4 casein fractions (α(S1), ß, α(S2), and κ) encoded by the 4 tightly linked autosomal genes (CSN1S1, CSN2, CSN1S2, and CSN3, respectively). In the present paper, we report for the first time a quantitative characterization of buffalo casein transcripts and show that the 4 genes are not transcribed and translated with the same efficiency. In particular, the analysis of individual milk samples obtained from 9 Mediterranean river buffaloes showed that the most abundant casein fractions were ß (53.45%) and α(S1) (20.61%), followed by α(S2) and κ, at 14.28 and 11.66%, respectively. Quantification of the corresponding mRNA showed that the percentage of transcripts of the 4 caseins was 16.48, 23.18, 55.87, and 4.47% for α(S1), ß, α(S2), and κ, respectively. Translation efficiency was 0.25 for CSN1S2, 1.31 for CSN1S1, 2.39 for CSN2, and 2.69 for the CSN3 transcripts, respectively. A comparison of nucleotide sequences with the Kozak consensus sequence was also carried out to investigate if the mRNA sequences might be responsible for the observed differences.

Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo.

Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P<0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products.

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.

X-Y sperm aneuploidy in 2 cattle (Bos taurus) breeds as determined by dual color fluorescent in situ hybridization (FISH).

The present study was undertaken to investigate aneuploidy rates in the sperm populations of 2 cattle (Bos taurus) breeds by using dual color fluorescent in situ hybridization (FISH) with Xcen and Y chromosome-specific painting probes, obtained by chromosome microdissection and DOP-PCR. Frozen semen from 10 Italian Friesian and 10 Italian Brown testing bulls was used for the investigation. For each bull, more than 5,000 sperm were analyzed, for a total of 52,586 and 51,342 sperm cells for the 2 breeds, respectively. The present study revealed - in both breeds - a preponderance of the Y-bearing sperm compared to the X-bearing sperm. Within each breed, a statistically significant variation in the various classes of aneuploidy (XX, YY and XY) was found: differences were found in the Friesian breed among the 3 diploidy classes, and in the Brown breed, among the 3 disomy classes (p < 0.05) as well as among the 3 diploidy classes (p < 0.01). However, the 2 breeds did not differ significantly in the overall mean rates of X-Y aneuploidy (disomy + diploidy) which amounts to 0.162% in the Italian Friesian and 0.142% in the Italian Brown. When meiosis I (MI) and II (MII) errors were compared, statistically significant differences (p < 0.01) were found in the disomy classes and in both breeds, whereas the differences between diploidy classes were not significant. Compared to humans, a lower level of aneuploidy has been found in the domestic species analyzed so far. The present study contributes to the establishment of a baseline level of aneuploidy in the sperm populations of 2 cattle breeds which could be used for monitoring future trends of reproductive health, especially in relation to environmental changes and mutagens.

The 450-band resolution G- and R-banded standard karyotype of the donkey (Equus asinus, 2n = 62).

Donkey chromosomes were earlier characterized separately by C-, G- and R-banding techniques. However, direct comparisons between G- and R-banding patterns have still not been carried out in this species. The present study reports this comparison at the 450-band level by using replication G- and R-banding patterns. Two sets of synchronized lymphocyte cultures were set up to obtain early (GBA+CBA-banding) and late (RBA-banding) BrdU incorporation. Slides were stained with acridine orange and observed under a fluorescence microscope. Reverse GBA+CBA- and RBA-banded karyotypes at the 450-band level were constructed. To verify G- and R-banding patterns in some acrocentric chromosomes, sequential GBA+CBA/Ag-NORs and RBA/Ag-NORs were also performed. The results of CBA-banding patterns obtained in 12 animals from 2 breeds showed a pronounced polymorphism of heterochromatin, especially in EAS1q-prox. Ideogrammatic representations of G- and R-banded karyotypes were constructed using only one common G- and R-banding nomenclature. In the present study both G- and R-banding patterns and relative ideograms are presented as standard karyotype for this species at the 450-band level.

Chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis).

The present study reports on the chromosomal expression and localization of aphidicolin-induced fragile sites in the standard karyotype of river buffalo (Bubalus bubalis, 2n = 50) with the aim of establishing a 'fragile site map' of the species. Totally, 400 aphidicolin-induced breakages were analyzed from eight young and clinically healthy animals, four males and four females; these breakages were localized in 106 RBG-negative chromosome bands or at the band-interband regions. The number of breakages per chromosome did not vary statistically 'among' the animals investigated but the differences among individual chromosomes were highly significant thus indicating that the chromosomal distribution of the breakages is not random and appears only partially related to chromosome length. Fragile sites were statistically determined as those chromosomal bands showing three or more breakages. In the river buffalo karyotype, 51 fragile sites were detected and localized on the standardized ideogram of the species. The most fragile bands were as follows: 9q213 with 24 breakages out of 400; 19q21 with 16, 17q21 and inacXq24 with 15, 15q23 with 13 and 13q23 with 12 breaks, respectively. Previous gene mapping analysis in this species has revealed that the closest loci to these fragile sites contain genes such as RASA1 and CAST (9q214), NPR3 and C9 (19q19), PLP and BTK (Xq24-q25), OarCP09 (15q24), and EDNRB (13q22) whose mutations are responsible for severe phenotypic malformations and immunodeficiency in humans as well as in mice and meat quality in pigs. Further cytogenetic and molecular studies are needed to fully exploit the biological significance of the fragile sites in karyotype evolution of domestic animals and their relationships with productive and reproductive efficiency of livestock.

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.

An SNP in the goat CSN2 promoter region is associated with the absence of beta-casein in milk.

So far, at least eight alleles in the goat CSN2 locus have been associated with the level of beta-casein expression in milk. Alleles CSN2(A), CSN2(A1), CSN2(B), CSN2(C), CSN2(D) and CSN2(E) have been associated with normal content (allele effects of about 5 g of beta-casein per litre), whereas the CSN2(0) and CSN2(01) alleles have been associated with non-detectable levels of beta-casein. Most of these alleles have been characterized genetically. Herein, we report the identification of a previously unreported SNP in the goat CSN2 promoter region (AJ011018:g.1311T>C), which is associated with the absence of beta-casein in the milk. Furthermore, we developed a PCR-based method that allows detection of this mutation.

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.

Cytogenetic damage and induction of pro-oxidant state in human lymphocytes exposed in vitro to gliphosate, vinclozolin, atrazine, and DPX-E9636.

We analyzed chromosome aberrations (CAs), sister chromatid exchanges (SCEs), mitotic index (MI), and glucose 6-phosphate dehydrogenase (G6PD) enzyme activity in human peripheral lymphocytes from three healthy donors exposed in vitro to different concentrations of gliphosate, vinclozolin, atrazine, and DPX-E9636. The pesticides gliphosate, vinclozolin, and atrazine have been studied in a broad range of genetic tests with predominantly conflicting or negative results, whereas little is known about the genotoxicity of DPX-E9636. In our experimental conditions, each chemical compound tested produced a dose-related increase in the percent of aberrant cells and an increase of SCE/cell. Furthermore, at the highest concentrations of vinclozolin, atrazine, and DPX-E9636, we observed a significant reduction of the mitotic index. The increase of G6PD activity in exposed lymphocyte cultures strongly indicated an induction of a pro-oxidant state of the cells as an initial response to pesticide exposure.

Measurement of micronuclei by cytokinesis-block method in bovine lymphocytes.

The present study was carried out in order to set up a standardized quantitative assay for spontaneous micronuclei in bovine lymphocytes. For this purpose the cytokinesis-block micronucleus (MN) method, originally proposed by Fenech and Morley (1985) for human lymphocytes, was applied to peripheral blood lymphocytes of 20 healthy cows of Italian Friesian breed. The results demonstrate that the optimal concentration of cytochalasin B to obtain the highest frequency of binucleated cells (mean = 400.26 +/- 23.76/1000 cells scored) was 6 micrograms/ml. The baseline frequency of spontaneous MN formation in 500 binucleated cells was 12.3 +/- 4.1, i.e., 3 times higher than that reported in human lymphocytes (Fenech and Morley, 1985; Scarfi et al., 1991). The possible reason(s) for this difference (sensitivity to cytochalasin B, chromosome number, environmental genotoxic pollutants) is discussed.

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.

Chromosome instability in a calf affected by congenital malformation.

High rates of structural chromosome aberrations were associated with increased yields of sister chromatid exchanges (SCE) in metaphase chromosomes of a new born female calf affected by a congenital malformation. The frequency of abnormal cells was 25 per cent in the abnormal calf, 8 per cent in its dam and 3 per cent in a group of four healthy cows. Chromatid and chromosome breaks were the most frequent types of chromosome aberration found in the malformed calf; centric fusions, chromosome fragments and deletions were much less common. The mean rate of SCE/cell in the malformed calf was nearly twice that of the control and the difference was statistically significant. Possible factors involved in the occurrence of such a malformation are discussed.