State of the art on the physical mapping of the Y-chromosome in the Bovidae and comparison with other species - A review.

The next generation sequencing has significantly contributed to clarify the genome structure of many species of zootechnical interest. However, to date, some portions of the genome, especially those linked to a heterogametic nature such as the Y chromosome, are difficult to assemble and many gaps are still present. It is well known that the fluorescence in situ hybridization (FISH) is an excellent tool for identifying genes unequivocably mapped on chromosomes. Therefore, FISH can contribute to the localization of unplaced genome sequences, as well as to correct assembly errors generated by comparative bioinformatics. To this end, it is necessary to have starting points; therefore, in this study, we reviewed the physically mapped genes on the Y chromosome of cattle, buffalo, sheep, goats, pigs, horses and alpacas. A total of 208 loci were currently mapped by FISH. 89 were located in the malespecific region of the Y chromosome (MSY) and 119 were identified in the pseudoautosomal region (PAR). The loci reported in MSY and PAR were respectively: 18 and 25 in Bos taurus, 5 and 7 in Bubalus bubalis, 5 and 24 in Ovis aries, 5 and 19 in Capra hircus, 10 and 16 in Sus scrofa, 46 and 18 in Equus caballus. While in Vicugna pacos only 10 loci are reported in the PAR region. The correct knowledge and assembly of all genome sequences, including those of genes mapped on the Y chromosome, will help to elucidate their biological processes, as well as to discover and exploit potentially epistasis effects useful for selection breeding programs.

TiO2-NPs and cadmium co-exposure: in vitro assessment of genetic and genomic DNA damage on Dicentrarchus labrax embryonic cells.

The increased titanium dioxide nanoparticles (TiO2-NPs) spread and their interaction with organic and inorganic pollutants arouses concern for the potential hazards for organisms and environment. This study tested in vitro the genotoxic effects of TiO2-NPs (1 µg/mL) and cadmium (Cd) (0.1 µg/mL) co-exposure using Dicentrarchus labrax embryonic cells (DLEC) as experimental model. The genotoxicity tests (Comet assay, Diffusion Assay and Random Amplification of Polymorphic DNA (RAPD-PCR) were conducted after 3, 24 and 48 hours of exposure to TiO2-NPs and Cd alone and in combination. The results showed that the percentage of DNA damage and apoptotic cells increases following 48 hours TiO2-NPs exposure, while DNA instability was detected for all the times tested. Cd induced genotoxic effects starting from 3 hour-exposure and for all the treatment times. Cd + TiO2-NPs co-exposure did not cause any genomic damage or apoptosis for all the exposure times. The possibility that Cd and TiO2-NPs form aggregates no longer able of penetrating the nucleus and damaging the genetic material is discussed.

Sperm Nuclei Analysis and Nuclear Organization of a Fertile Boar-Pig Hybrid by 2D FISH on Both Total and Motile Sperm Fractions.

A wide range of mammalian hybrids has recently been found by chance or through population-screening programs, but studies about their fertilizing capacity remain scarce and incomplete. Most of them are assumed to be sterile due to meiotic arrest caused by the failure of chromosome pairings. In this study, we evaluated both sperm meiotic segregation, by 2D fluorescent in situ hybridization (FISH) analysis, and sperm quality (Sperm Chromatin Structure Assay) by flow cytometer in a fertile boar-pig hybrid (2n = 37,XY) originating from a Nero Siciliano pig breed (Sus scrofa domesticus) and a wild boar (Sus scrofa ferus). Spermatozoa were also separated by a dual-layer (75-60%) discontinuous Percoll gradient, resulting in two fractions with a significantly better overall quality in the motile sperm fraction. These data were confirmed by FISH analysis also, where the frequencies of spermatozoa with a regular chromosome composition were 27% in total sperm fraction and 64% in motile sperm fraction. We also evaluated the nuclear architecture in all counted spermatozoa, showing a chromatin distribution changing when chromosome abnormalities occur. Our results demonstrate that the chromosome pairing has a minimal effect on the sperm segregation and semen quality of a boar-pig hybrid, making it fertile and harmful for the conservation of autochthonous pig breeds.

A de novo reciprocal chromosomal translocation t(3;6)(p14;q26) in the black Lucano pig.

In the past two decades, several cytogenetic screening programmes identified different chromosome rearrangements in pig, most of which represented by reciprocal translocation (rcp). This chromosome abnormality does not involve the variation in the number of chromosomes, but only the rearrangement of genetic material, resulting in phenotypically normal carriers with fertility problems. During an occasional cytogenetic screening, a new reciprocal translocation was detected in the black Lucano pig native breed. We analysed 15 animals reared by a family-run piggery in Basilicata region (Southern Italy). After karyotyping, four pigs (two boars and two sows) revealed two unpaired chromosomes. Analysis of the RBA karyotype and the dual-colour FISH technique confirmed that these pigs showed the same reciprocal translocation involving the chromosomes SSC3 and SSC6. The precise location of breakpoints was identified by RBH-FISH t(3;6)(p14;q26), whereas the analysis of the pedigree showed a case of Mendelian inheritance within a family, after the de novo occurrence of the new rcp. Considering the consequences of the rcp on the fertility, this study points out the importance of the cytogenetic screening in the native breeds for the safeguard of the genetic biodiversity and the sustainability of the rural areas.

Clinical, cytogenetic and molecular genetic characterization of a tandem fusion translocation in a male Holstein cattle with congenital hypospadias and a ventricular septal defect.

Hypospadias, disorder of sex development (DSD), is a sporadic congenital abnormality of the genital region in male ruminants, which is characterized by a non-fused urethra during fetal development. Detailed clinical examination classified the hypospadias phenotype of a male Holstein calf studied here as the perineal type. In combined use of cytogenetic analysis and whole genome sequencing, a non-mosaic, pseudo-monosomy 59, XY + tan(18;27) was detected. This chromosomal aberration had its origin in a tandem fusion translocation of the bovine autosomes (BTA) 18 and 27 with an accompanying loss of genomic sequences mainly in the distal end of BTA 18 and the proximal end of BTA 27. The resulting phenotype included hypospadias, growth retardation and ventricular septal defect.

Cytogenetic tests reveal no toxicity in lymphocytes of rabbit (Oryctolagus cuniculus, 2n=44) feed in presence of verbascoside and/or lycopene.

Phenylpropanoid glycosides (PPG), like other phenolic compounds, are a powerful antioxidants and the Verbascoside (VB) is one of the most active of them. A previous study, by using in vitro exposure of blood human lymphocytes to Verbascoside, reported a significant increasings of chromosome fragility compared to control. In the present study, four homogeneous groups of rabbits were used to test in vivo the VB and/or Lycopene (LP) by feeding the animals without VB and LP (control), in presence of VB or/and LP for 80 days. Lymphocyte cell cultures were performed in three different times: 0, 40 and 80 days of the experiment and the cytogenetic tests that we used [CA-test (Chromosome Abnormalities in terms of chromosome and chromatid breaks) and Sister Chromatid Exchange (SCE-test)] have revealed no mutagenic effects on chromosomes. Indeed, mean values/cell of CA and SCE decreased during the experiment with some difference among and within groups, with significant decreasing value only for some group. The study shows clear evidence that diets rich in Verbascoside (and/or Lycopene) do not originate any mutagenic activity, resulting no cytotoxic for the animals and, suggesting a possible their use in both animal and human diets.

Sister chromatid exchange test in river buffalo lymphocytes treated in vitro with furocoumarin extracts.

Furocoumarin extracts from Psoralea morisiana, the endemic Sardinian legume species, were tested for their mutagenic potential on river buffalo blood cells. The results obtained performing the sister chromatid exchange (SCE) test in blood cultures of five river buffalo calves (exposure to furocoumarins for 72h) and five cows (exposure to furocoumarins for 3h, in the absence and presence of S9 metabolic activator) are reported. Significant differences in mean values of SCEs were observed in cells of calves compared to control cells (unexposed), but no differences in SCE mean values were found between treated and untreated cells of cows in the presence or absence of S9. SCE mean values were much higher in cells of cows (exposed and control) than in cells of calves. Indeed, in calf cells, SCE mean values/cell (±SD) were 6.66±2.45 in the control and 7.63±3.01, 9.03±3.90, 9.53±3.60 and 9.99±3.41 in treated cells at 50, 100, 200 and 400 µg/ml of furocoumarin extracts, respectively. In cow cells, grown in presence of S9, SCE mean values/cell were 11.49±4.78 and 11.65±5.19 in treated cells at 100 and 200 µg/ml of furocoumarins and 11.66±5.45 in the control. In cow cells grown in absence of S9, SCE mean values were 11.81±6.14 in the control and 12.35±7.09 and 12.01±5.43, respectively, in the presence of 100 and 200 µg/ml of furocoumarins. Despite their higher SCE values in the absence of S9, no statistically significant differences were found when these values were compared with those shown in presence of S9, suggesting no mutagenic action of furocoumarins in cows, at the doses used in this study.

Fatal Outcome in a Newborn Calf Associated with Partial Trisomy 25q and Partial Monosomy 11q, 60,XX,der(11)t(11;25)(q11;q14∼21).

A newborn calf of the Agerolese cattle breed underwent clinical cytogenetic investigation because of hyperflexion of the forelimbs, red eyes and the inability to stand. Anamnesis revealed that the mother, phenotypically normal, carried a chromosomal aberration. The newborn died after 2 weeks, and no remarkable alterations were found by the veterinarian on postmortem examination. The mother was a carrier of a reciprocal balanced translocation rcp(11;25)(q11,q14∼21) detected after a cytogenetic investigation in 2011; however, the analysis of the newborn revealed a different chromosomal aberration with partial trisomy of chromosome 25 and partial monosomy of chromosome 11. In fact, the results showed both chromosomes 25, one chromosome 11 and only one long derivative chromosome (der11). FISH analysis, performed using BAC clones, confirmed the chromosomes and their regions involved. Finally, both the localization of the breakpoints on band q11 (centromere) of chromosome 11 and band q14-21 of chromosome 25, and the complete loss of the der25 identified the aberration as an unbalanced translocation 60,XX,der(11)t(11;25)(q11;q14∼21). A comparison with human chromosomes was also performed to search for similarities and possible genes involved in order to study their effects, thus extending the knowledge of these aberrations by case reports.

Analysis of chromosome damage by sister chromatid exchange (SCE) and redox homeostasis characterization on sheep flocks from Sardinian pasturelands.

Over the last decades, an increase of pollutants of diverse origin (industrial, military, mining, etc.) was recorded in several areas of Sardinia Island. We report the results of a multidisciplinary and complementary study based on cytogenetic and physiological analyses. The data obtained show the effects of the environmental impact on six sheep flocks (Sardinian breed) grazing on natural pasturelands next to possible polluted areas and compared to three herds grazing in different areas far from those potentially contaminated and used as control. Sister chromatid exchange (SCE) test was used as cytogenetic test to analyze chromosomal damages and it was performed on peripheral blood samples collected from 129 adult sheep (age > 4 years) randomly selected from polluted (92 animals) and control (37 animals) areas. Two types of cell cultures were performed: without (normal cultures) and with the addition of 5-BrdU. SCE-mean values estimated over 35 cells counted for each animal were 8.65 ± 3.40, 8.10 ± 3.50, 8.05 ± 3.08, 7.42 ± 3.34, 9.28 ± 3.56 and 8.38 ± 3.29 in the exposed areas, whereas the average values were 7.86 ± 3.31 in the control group. Significant increases (P < 0.01) of SCEs were found in three investigated areas of Southern Sardinia. Furthermore, sheep of the same flocks were characterized for blood redox homeostasis in order to define the potential targets of oxidative damage and to identify biomarkers of the extent of animal exposure to environmental contaminants. The plasma levels of Asc, Toc and Ret were found to be significantly lower (P < 0.001) in exposed sheep (I, II, IV and V) than in the control group. TAC as well as GPx and SOD activities were higher in control than in the exposed groups (P < 0.001). Finally, plasma levels of N-Tyr, PC, and LPO were significantly lower (P < 0.001) in the control group than in the exposed groups.

Sequential cross-species chromosome painting among river buffalo, cattle, sheep and goat: a useful tool for chromosome abnormalities diagnosis within the family Bovidae.

The main goal of this study was to develop a comparative multi-colour Zoo-FISH on domestic ruminants metaphases using a combination of whole chromosome and sub-chromosomal painting probes obtained from the river buffalo species (Bubalus bubalis, 2n = 50,XY). A total of 13 DNA probes were obtained through chromosome microdissection and DOP-PCR amplification, labelled with two fluorochromes and sequentially hybridized on river buffalo, cattle (Bos taurus, 2n = 60,XY), sheep (Ovis aries, 2n = 54,XY) and goat (Capra hircus, 2n = 60,XY) metaphases. The same set of paintings were then hybridized on bovine secondary oocytes to test their potential use for aneuploidy detection during in vitro maturation. FISH showed excellent specificity on metaphases and interphase nuclei of all the investigated species. Eight pairs of chromosomes were simultaneously identified in buffalo, whereas the same set of probes covered 13 out 30 chromosome pairs in the bovine and goat karyotypes and 40% of the sheep karyotype (11 out of 27 chromosome pairs). This result allowed development of the first comparative M-FISH karyotype within the domestic ruminants. The molecular resolution of complex karyotypes by FISH is particularly useful for the small chromosomes, whose similarity in the banding patterns makes their identification very difficult. The M-FISH karyotype also represents a practical tool for structural and numerical chromosome abnormalities diagnosis. In this regard, the successful hybridization on bovine secondary oocytes confirmed the potential use of this set of probes for the simultaneous identification on the same germ cell of 12 chromosome aneuploidies. This is a fundamental result for monitoring the reproductive health of the domestic animals in relation to management errors and/or environmental hazards.

The utility of chromosome microdissection in clinical cytogenetics: a new reciprocal translocation in sheep.

Local sheep breeders and scientists in Italy cooperate and conduct research on the genetic improvement of autochthonous genetic types (AGTs) by various approaches, including a cytogenetic breeding selection since 2011. The Laticauda sheep (Ovis aries, 2n = 54) breed is one of the AGTs reared in the Campania region (southern Italy). Performing cytogenetic analyses, we have detected and described a novel reciprocal translocation in a Laticauda sheep identified as 54,XX t(18;23)(q14;q26). Our data support recurring appeals that suggest the regular performance of cytogenetic analyses for monitoring genetic health of livestock species. In total, 5 cases of reciprocal translocations in sheep are known, including the new case. None of them has any phenotypic effect on the living offspring. However, affected animals are characterized by sterility or have a low fertility which can have an effect on breeding success and on economical balance. Presence and kind of the described novel chromosomal aberration were detected by performing CBA-banding and FISH mapping with telomeric probes. RBA-banding allowed the karyotyping of sheep chromosomes and the identification of aberrant chromosomes and regions involved in the new reciprocal translocation. Whole chromosome painting (WCP) probes received from equivalent chromosomes in cattle and the derivative sheep chromosome 18 confirmed the cytogenetic data. This way, our study underlined both the importance of WCP probes by chromosome microdissection and a new way to use WCP probes directly generated from derivative chromosomes.

Development of a sequential multicolor-FISH approach with 13 chromosome-specific painting probes for the rapid identification of river buffalo (Bubalus bubalis, 2n = 50) chromosomes.

The development of new molecular techniques (array CGH, M-FISH, SKY-FISH, etc.) has led to great advancements in the entire field of molecular cytogenetics. However, the application of these methods is still very limited in farm animals. In the present study, we report, for the first time, the production of 13 river buffalo (Bubalus bubalis, 2n = 50) chromosome-specific painting probes, generated via chromosome microdissection and the DOP-PCR procedure. A sequential multicolor-FISH approach is also proposed on the same slide for the rapid identification of river buffalo chromosome/arms, namely, 1p-1q, 2p-2q, 3p-3q, 4p-4q, 5p-5q, 18, X, and Y, using both conventional and late-replicating banded chromosome preparations counterstained by DAPI. The provided 'bank' of chromosome-specific painting probes is useful for any further cytogenetic investigation not only for the buffalo breeds, but also for other species of the family Bovidae, such as cattle, sheep, and goats, for chromosome abnormality diagnosis, and, more generally, for evolutionary studies.

Genomic analysis of cattle rob(1;29).

Robertsonian translocation (rob) involving chromosomes 1 and 29 represents the most frequent chromosome abnormality observed in cattle breeds intended for meat production. The negative effects of this anomaly on fertility are widely demonstrated, and in many countries, screening programs are being carried out to eliminate carriers from reproduction. Although rob(1;29) was first observed in 1964, the genomic structure of this anomaly is partially unclear. In this work, we demonstrate that, during the fusion process, around 5.4 Mb of the pericentromeric region of BTA29 moves to the q arm, close to the centromere, of rob(1;29). We also clearly show that this fragment is inverted. We find that no deletion/duplication involving sequences reported in the BosTau6 genome assembly occurred during the fusion process which originates this translocation.

Fluorescence in situ hybridization mapping of six loci containing genes involved in the dioxin metabolism of domestic bovids.

Six loci containing genes involved in the dioxin metabolism (ARNT, AHR, CYP1A1, CYP1A2, CYP1B1 and AHRR) were assigned, for the first time, to cattle (Bos taurus, 2n = 60, BTA), river buffalo (Bubalus bubalis, 2n = 50, BBU), sheep (Ovis aries, 2n = 54, OAR) and goat (Capra hircus, 2n = 60, CHI) chromosomes by comparative FISH-mapping and R-banding using bovine BAC-clones. The following chromosome locations were found: ARNT to BTA3q21, BBU6q21, OAR1p21 and CHI3q21, AHR to BTA4q15, BBU8q15, OAR4q15 and CHI4q15; CYP1A1 and CYP1A2 to BTA21q17, BBU20q17, OAR18q17 and CHI21q17; CYP1B1 to BTA11q16, BBU12q22, OAR3p16 and CHI11q16, AHRR to BTA20q24, BBU19q24, OAR16q24 and CHI20q24. All loci were mapped at the same homoeologous chromosomes and chromosome bands of the four bovid species. Comparisons with corresponding human locations were also reported.