Effect of the rob(1;29) translocation on the fertility of beef cattle reared under extensive conditions: A 30-year retrospective study.

The Robertsonian translocation 1/29 (rob(1;29)) is the most worldwide widespread chromosomal abnormality in domestic animals. Previous studies have demonstrated its negative effect on fertility in dairy herds, but not in beef cattle extensively bred. In this study, we analysed the effect of rob(1;29) in a Retinta cattle breed data set gathered during the last 30 years. The data presented herein include rob(1;29) analysis of 11,505 cows from 251 herds, pedigree information of 24,790 animals and 67,457 calving records. Fertility was evaluated using estimated breeding values for the reproductive efficiency (Re), calculated as the percentage ratio between the number of calvings of an individual and the number expected in an optimal situation. Our results showed that cows carrying the heterozygote genotype showed a significant decrease in their Re (-5.10%, p < .001). No decrease was detected in free rob(1;29) animals and homozygous carriers. In addition, the incidence of rob(1;29) in the breed fertility was decreased to very low values after 30 years of avoiding selection of bulls' carrier as stallions. The effect of rob(1;29) on cattle fertility is only significant when the prevalence of carrier individuals is high. Selecting against the disease only by the paternal side reduced the incidence to negligible values.

Molecular mechanisms and topological consequences of drastic chromosomal rearrangements of muntjac deer.

Muntjac deer have experienced drastic karyotype changes during their speciation, making it an ideal model for studying mechanisms and functional consequences of mammalian chromosome evolution. Here we generated chromosome-level genomes for Hydropotes inermis (2n = 70), Muntiacus reevesi (2n = 46), female and male M. crinifrons (2n = 8/9) and a contig-level genome for M. gongshanensis (2n = 8/9). These high-quality genomes combined with Hi-C data allowed us to reveal the evolution of 3D chromatin architectures during mammalian chromosome evolution. We find that the chromosome fusion events of muntjac species did not alter the A/B compartment structure and topologically associated domains near the fusion sites, but new chromatin interactions were gradually established across the fusion sites. The recently borne neo-Y chromosome of M. crinifrons, which underwent male-specific inversions, has dramatically restructured chromatin compartments, recapitulating the early evolution of canonical mammalian Y chromosomes. We also reveal that a complex structure containing unique centromeric satellite, truncated telomeric and palindrome repeats might have mediated muntjacs' recurrent chromosome fusions. These results provide insights into the recurrent chromosome tandem fusion in muntjacs, early evolution of mammalian sex chromosomes, and reveal how chromosome rearrangements can reshape the 3D chromatin regulatory conformations during species evolution.

Screening and detection of chromosomal copy number alterations in the domestic horse using SNP-array genotyping data.

Chromosomal abnormalities are a common cause of infertility in horses. However, they are difficult to detect using automated methods. Here, we propose a simple methodology based on single nucleotide polymorphism (SNP)-array data that allows us to detect the main chromosomal abnormalities in horses in a single procedure. As proof of concept, we were able to detect chromosomal abnormalities in 33 out of 268 individuals, including monosomies, chimerisms, and male and female sex-reversions, by analyzing the raw signal intensity produced by an SNP array-based genotyping platform. We also demonstrated that the procedure is not affected by the SNP density of the array employed or by the inbreeding level of the individuals. Finally, the methodology proposed in this study could be performed in an open bioinformatic environment, thus permitting its integration as a flexible screening tool in diagnostic laboratories and genomic breeding programs.

Evaluation of the genotoxic and cytotoxic effects of exposure to the herbicide 2,4-dichlorophenoxyacetic acid in Astyanax lacustris (Pisces, Characidae) and the potential for its removal from contaminated water using a biosorbent.

The genotoxic and cytotoxic effects of 2,4-dichlorophenoxyacetic acid (2,4-D) on specimens of Astyanax lacustris were evaluated using different biomarkers. Additionally, this study evaluated the efficiency of an activated carbon filter made from the husks green coconut, which was used as a biosorbent to remove 2,4-D dissolved in the water, and the potential effectiveness of this procedure for the reduction of the toxic effects of this compound on A. lacustris. Three sublethal concentrations of 2,4-D (10, 20, and 40 mg L-1) were tested over 24, 48, and 72 h, and their effects on Astyanax lacustris were evaluated using chromosomal aberration test, the mitotic index, the frequency of micronuclei and nuclear alterations, and the comet assay. Exposure to 2,4-D increased the frequency of chromosomal aberrations, reduced the mitotic index, and caused significant levels of nuclear modification in some of the treatments, in comparison with the negative control. The comet assay revealed DNA damage (classes 1-3) at all 2,4-D concentrations, reaching significant levels in the 20 mg L-1 (48 h) and 40 mg L-1 (72 h) treatments. The coconut husk biosorbent was highly effective for the removal of 2,4-D and the fish exposed to the water decontaminated by this filter had low levels of cellular alteration. The findings of the present study demonstrated, for the first time, the genotoxic and cytotoxic effects of 2,4-D in Astyanax lacustris, as well as suggests the potential application of a biosorbent for the effective decontamination of water contaminated with pesticides.

Cytogenetic analysis of nuclear abnormalities in the erythrocytes of gecko lizards (Phyllopezus periosus) collected in a semi-arid region of northeast Brazil: Possible effects of natural background radioactivity.

High natural-background radioactivity levels occur in the semi-arid region of the State of Rio Grande do Norte, northeastern Brazil. We have studied the lizard Phyllopezus periosus, an endemic species of the Brazilian caatinga with saxicolous habitat, as a bioindicator of environmental quality. Specimens were collected in three areas, an environmental protection area and two areas recognized as having high natural background radiation, one of these being a mining area. Level of metals and gamma radiation emitters present in the water sources potentially used by the lizards were measured. The biological endpoints assessed were micronuclei and nuclear abnormalities in blood samples. Significant differences in background radioactivity levels were found among the assessed areas. Statistically significant differences in micronuclei and nuclear abnormality frequencies were seen, among the study areas and a relationship between radioactivity level and genetic damage was observed.

Determination of cytogenetic markers for biological monitoring in coypu (Myocastor coypu).

Cytogenetic tests are used to assess the influence of physical and chemical factors with potential mutagenic and genotoxic properties on the animal organism. The test results make it possible to eliminate mutagens, as well as helping predict possible genetic consequences in animal cells and assess animal resistance. The aim of this study was to examine, using cytogenetic tests, the spontaneous chromosome and DNA damage in coypu lymphocytes. Four tests: fragile site (FS), bleomycin (BLM), micronucleus, (MN) and comet were used for the first time in coypu cells. The averages with standard deviations obtained in the research were as follows: 3.30 ± 0.80 fragile sites/cell; 0.63 ± 0.80 BLM damage/cell; 6.10 ± 0.53% binucleated cells with MN; and 3.24 ± 0.63% DNA in tail. The present analysis showed high interindividual variation in spontaneous chromosomal and DNA damage levels. In the case of micronucleus, fragile sites, and comet assays, the differences between animals were statistically significant. The data suggest that these assays are sensitive enough to detect some effects on an individual animal and can be proposed as tools for coypu biomonitoring.

Selection based on morphological features of porcine embryos produced by in vitro fertilization: Timing of early cleavages and the effect of polyspermy.

The aim of this study was to examine whether a morphological approach is efficient for selecting high-quality porcine embryos produced by in vitro fertilization (IVF) under high polyspermy conditions. Frozen-thawed Meishan epididymal spermatozoa showing moderate and high polyspermy were subjected to IVF (1 × 105  sperms/ml). Under conditions of moderate polyspermy, 4-cell embryos selected at 48 hr after IVF (single selection) and 8-cell embryos selected at 79 hr after IVF from the collected 4-cell embryos (double selection) showed high developmental competence. Likewise, 4- and 8-cell embryos produced by IVF under high polyspermy conditions also showed high competence for development to blastocysts. However, blastocysts derived from high polyspermy conditions had significantly fewer cells than those produced under moderate polyspermy conditions. Furthermore, the frequency of nuclear and chromosomal abnormalities in 4- and 8-cell embryos produced under conditions of high polyspermy was significantly (p < .05) higher in comparison to moderate polyspermy conditions. These findings suggest that although high polyspermy affects the frequency of nuclear and chromosomal anomalies in porcine IVF embryos, subsequent selection based on morphological features of 4- and 8-cell embryos even under high polyspermy conditions, could be an alternative option for selecting porcine IVF embryos with high development ability.

Ascertaining the paternal lineage in crossbred calves.

Karyotyping is one among the culling parameter used for taking up culling decisions. Cytogenetic screening of breeding bulls has been recommended to screen for chromosomal abnormalities before semen production in artificial insemination. The chromosomal analysis of a Holstein Friesian crossbred bull revealed the presence of acrocentric Y-chromosome, which was further confirmed by CBG banding. The shape of the Y-chromosome determining that male line used for crossbreeding was from indigenous origin. Karyotyping is a best and reliable technique for the identification of crossbred calves born to the indigenous bulls.

Integrated analysis of transcriptome, methylome and copy number aberrations data of marginal zone lymphoma and follicular lymphoma in dog.

Marginal zone lymphoma (MZL) and follicular lymphoma (FL) are classified as indolent B-cell lymphomas in dogs. Aside from the clinical and histopathological similarities with the human counterpart, the molecular pathogenesis remains unclear. We integrated transcriptome, genome-wide DNA methylation and copy number aberration analysis to provide insights on the pathogenesis of canine MZL (n = 5) and FL (n = 7), also comparing them with diffuse large B-cell lymphoma (DLBCL). Transcriptome profiling highlighted the presence of similar biological processes affecting both histotypes, including BCR and TLR signalling pathways. However, FLs showed an enrichment of E2F targets, whereas MZLs were characterized by MYC-driven transcriptional activation signatures. FLs showed a distinctive loss on chr1 containing CEACAM23 and 24, conversely MZLs presented multiple recurrent gains on chr13, where MYC is located. The distribution of methylation peaks was similar between the two histotypes. Integrating data from the three omics, FLs resulted clearly separated from MZLs and DLBCL dataset. MZLs showed the enrichment of FoxM1 network and TLR associated TICAM1-dependent IRFs activation pathway. However, no specific signatures differentiated MZLs from DLBCLs. In conclusion, our study presents the first comprehensive analysis of molecular and epigenetic pathogenesis of canine FL and MZL.

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.

Prognostic value of somatic focal amplifications on chromosome 30 in canine oral melanoma.

Canine oral melanoma is the first malignancy of the oral cavity in dogs and is characterized by a local invasiveness and a high metastatic propensity. A better knowledge of genetic alterations is expected to improve management of this tumour. Copy number alterations are known characteristics of mucosal melanomas both in dogs and humans. The goal of this study was to explore the prognostic value of somatic focal amplifications on chromosomes (Canis Familiaris [CFA]) 10 and 30 in canine oral melanoma. The cohort included 73 dogs with oral melanoma confirmed by histology, removed surgically without adjuvant therapy and with a minimal follow-up of 6 months. Epidemiological, clinical and histological data were collected and quantitative-PCR were performed on formalin-fixed paraffin-embedded (FFPE) samples to identify specific focal amplifications. The 73 dogs included in the study had a median survival time of 220 days. Focal amplifications on CFA 10 and 30 were recurrent (49.3% and 50.7% of cases, respectively) and CFA 30 amplification was significantly associated with the amelanotic phenotype (P = .046) and high mitotic index (MI; P = .0039). CFA 30 amplification was also linked to poor prognosis (P = .0005). Other negative prognostic factors included gingiva location (P = .003), lymphadenomegaly (P = .026), tumour ulceration at diagnosis (P = .003), MI superior to 6 mitoses over 10 fields (P = .001) and amelanotic tumour (P = .029). In multivariate analyses using Cox proportional hazards regression, CFA 30 amplification (Hazard ratio [HR] = 2.08; P = .011), tumour location (HR = 2.20; P = .005) and histological pigmentation (HR = 1.87; P = .036) were significantly associated with shorter survival time. Focal amplification of CFA 30 is linked to an aggressive subset and constitutes a new prognostic factor.

In vitro production of horse embryos predisposes to micronucleus formation, whereas time to blastocyst formation affects likelihood of pregnancy.

Invitro embryo production is an increasingly popular means of breeding horses. However, success is limited by a high incidence of early embryo loss. Although there are various possible causes of pregnancy failure, chromosomal abnormalities, including aneuploidy, are important potential contributors. This study evaluated the frequency of micronucleus formation as a proxy for aneuploidy in invitro-produced (IVP) and invivo-derived horse blastocysts. Associations between IVP embryo morphology, frequency of nuclear abnormalities and the likelihood of pregnancy were investigated. IVP blastocysts exhibited a higher frequency of cells with micronuclei than invivo-derived embryos (10% vs 1% respectively; P=0.05). This indication of chromosomal instability may explain the higher incidence of pregnancy failure after transfer of IVP embryos. However, the frequency of micronuclei was not correlated with brightfield microscopic morphological characteristics. Nevertheless, IVP embryos reaching the blastocyst stage after Day 9 of invitro culture were less likely to yield a pregnancy than embryos that developed to blastocysts before Day 9 (27% vs 69%), and embryos that had expanded before transfer were more likely to undergo embryonic death than those that had not expanded (44% vs 10%). These findings indicate that current embryo culture conditions are suboptimal and that the speed of embryo development is correlated with pregnancy survival.

Prevalence of chromosomal aberrations in breeding pigs in Spain.

The main aim of this study was to document the prevalence of chromosomal aberrations found to date on the pig population in Spain, a country in which this production sector has a critical role, being the fourth country in the world in pig production and the second one within the European Union. The total number of animals studied was 849, and the founded frequency of carrier pigs with chromosomal alterations was 3.8%. When only the structural alterations were considered, the prevalence in males was 3.3%. This percentage is far from the 0.5% of carrier boars that has been estimated in France, a country where there is a systematic cytogenetic screening of future breeding pigs since 1992. In order to avoid the productive and economic losses caused by karyotype alterations in breeding pigs, it would be important to establish a cytogenetic screening of breeding animals at artificial insemination centres and genetic selection farms.

Prevalence and consequences of chromosomal abnormalities in Canadian commercial swine herds.

BACKGROUND: Structural chromosome abnormalities are well known as factors that reduce fertility rate in domestic pigs. According to large-scale national cytogenetic screening programs that are implemented in France, it is estimated that new chromosome abnormalities occur at a rate of 0.5 % in fertility-unproven boars. RESULTS: This work aimed at estimating the prevalence and consequences of chromosome abnormalities in commercial swine operations in Canada. We found pig carriers at a frequency of 1.64 % (12 out of 732 boars). Carrier pigs consistently showed lower fertility values. The total number of piglets born for litters from carrier boars was between 4 and 46 % lower than the herd average. Similarly, carrier boars produced litters with a total number of piglets born alive that was between 6 and 28 % lower than the herd average. A total of 12 new structural chromosome abnormalities were identified. CONCLUSIONS: Reproductive performance is significantly reduced in sires with chromosome abnormalities. The incidence of such abnormal sires appears relatively high in populations without routine cytogenetic screening such as observed for Canada in this study. Systematic cytogenetic screening of potential breeding boars would minimise the risk of carriers of chromosome aberrations entering artificial insemination centres. This would avoid the large negative effects on productivity for the commercial sow herds and reduce the risk of transmitting abnormalities to future generations in nucleus farms.

Chromosome Aberrations and Fertility Disorders in Domestic Animals.

The association between chromosomal abnormalities and reduced fertility in domestic animals is well recorded and has been studied for decades. Chromosome aberrations directly affect meiosis, gametogenesis, and the viability of zygotes and embryos. In some instances, balanced structural rearrangements can be transmitted, causing fertility problems in subsequent generations. Here, we aim to give a comprehensive overview of the current status and future prospects of clinical cytogenetics of animal reproduction by focusing on the advances in molecular cytogenetics during the genomics era. We describe how advancing knowledge about animal genomes has improved our understanding of connections between gross structural or molecular chromosome variations and reproductive disorders. Further, we expand on a key area of reproduction genetics: cytogenetics of animal gametes and embryos. Finally, we describe how traditional cytogenetics is interfacing with advanced genomics approaches, such as array technologies and next-generation sequencing, and speculate about the future prospects.

Effect of post-thaw addition of seminal plasma on motility, viability and chromatin integrity of cryopreserved donkey jack (Equus asinus) spermatozoa.

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen-thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post-thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP-αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post-thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post-thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.

Novel tools for characterising inter and intra chromosomal rearrangements in avian microchromosomes.

Avian genome organisation is characterised, in part, by a set of microchromosomes that are unusually small in size and unusually large in number. Although containing about a quarter of the genome, they contain around half the genes and three quarters of the total chromosome number. Nonetheless, they continue to belie analysis by cytogenetic means. Chromosomal rearrangements play a key role in genome evolution, fertility and genetic disease and thus tools for analysis of the microchromosomes are essential to analyse such phenomena in birds. Here, we report the development of chicken microchromosomal paint pools, generation of pairs of specific microchromosome BAC clones in chicken, and computational tools for in silico comparison of the genomes of microchromosomes. We demonstrate the use of these molecular and computational tools across species, suggesting their use to generate a clear picture of microchromosomal rearrangements between avian species. With increasing numbers of avian genome sequences that are emerging, tools such as these will find great utility in assembling genomes de novo and for asking fundamental questions about genome evolution from a chromosomal perspective.

Karyotype divergence and spreading of 5S rDNA sequences between genomes of two species: darter and emerald gobies ( Ctenogobius , Gobiidae).

Karyotype analyses of the cryptobenthic marine species Ctenogobius boleosoma and C. smaragdus were performed by means of classical and molecular cytogenetics, including physical mapping of the multigene 18S and 5S rDNA families. C. boleosoma has 2n = 44 chromosomes (2 submetacentrics + 42 acrocentrics; FN = 46) with a single chromosome pair each carrying 18S and 5S ribosomal sites; whereas C. smaragdus has 2n = 48 chromosomes (2 submetacentrics + 46 acrocentrics; FN = 50), also with a single pair bearing 18S rDNA, but an extensive increase in the number of GC-rich 5S rDNA sites in 21 chromosome pairs. The highly divergent karyotypes among Ctenogobius species contrast with observations in several other marine fish groups, demonstrating an accelerated rate of chromosomal evolution mediated by both chromosomal rearrangements and the extensive dispersion of 5S rDNA sequences in the genome.

A tortoiseshell male cat: chromosome analysis and histologic examination of the testis.

Tortoiseshell coat color is normally restricted to female cats due to X-linkage of the gene that encodes the orange coat color. Tortoiseshell male cats do, however, occur at a low frequency among tortoiseshell cats because of chromosome aberrations similar to the Klinefelter syndrome in man: the extra X chromosome of a 39,XXY karyotype introduces the possibility of an orange and a non-orange allele which produce the mixture of orange and non-orange coat spotting known as tortoiseshell. We analyzed the chromosome complement of a fibroblast culture and did histological examinations of testicular tissue from a tortoiseshell male cat referred to us. Chromosome analysis using RBA-banding consistently revealed a 39,XXY karyotype. Histological examinations of testis biopsies from this cat showed degeneration of the tubules, hyperplasia of the interstitial tissue, and complete loss of germ cells. Immunostaining using anti-vimentin and anti-VASA (DDX4) showed that only Sertoli cells and no germ cells were observed in the testicular tubules. As no sign of spermatogenesis was detected, we conclude that this is a classic case of a sterile, male tortoiseshell cat with a 39,XXY chromosome complement.

Ovarian dysgenesis in an alpaca with a minute chromosome 36.

A 4-year-old female alpaca (Lama pacos [LPA]) was presented to the Oregon State Veterinary Teaching Hospital for failure to display receptive behavior to males. Although no abnormalities were found on physical examination, transrectal ultrasonographic examination of the reproductive tract revealed uterine hypoplasia and ovarian dysgenesis. Cytogenetic analysis demonstrated a normal female 74,XX karyotype with 1 exceptionally small (minute) homologue of autosome LPA36. Chromosome analysis by Giemsa staining and DAPI- and C-banding revealed that the minute LPA36 was submetacentric, AT-rich, and largely heterochromatic. Because of the small size and lack of molecular markers, it was not possible to identify the origin of the minute. There is a need to improve molecular cytogenetic tools to further study the phenomenon of this minute chromosome and its relation to female reproduction in alpacas and llamas.