Genetic diagnosis of sex chromosome aberrations in horses based on parentage test by microsatellite DNA and analysis of X- and Y-linked markers

REASONS FOR PERFORMING STUDY: Sex chromosome aberrations are often associated with clinical signs that affect equine health and reproduction. However, abnormal manifestation with sex chromosome aberration usually appears at maturity and potential disorders may be suspected infrequently. A reliable survey at an early stage is therefore required. OBJECTIVES: To detect and characterise sex chromosome aberrations in newborn foals by the parentage test and analysis using X- and Y-linked markers. METHODS: We conducted a genetic diagnosis combined with a parentage test by microsatellite DNA and analysis of X- and Y-linked genetic markers in newborn light-breed foals (n = 17, 471). The minimum incidence of sex chromosome aberration in horses was estimated in the context of available population data. RESULTS: Eighteen cases with aberrations involving 63,XO, 65,XXY and 65,XXX were found. The XO, XXY (pure 65,XXY and/or mosaics/chimaeras) and XXX were found in 0.15, 0.02 and 0.01% of the population, respectively, based solely on detection of abnormal segregation of a single X chromosome marker, LEX003. CONCLUSIONS AND POTENTIAL RELEVANCE: Detection at an early age and understanding of the prevalence of sex chromosome aberrations should assist in the diagnosis and managment of horses kept for breeding. Further, the parental origin of the X chromosome of each disorder could be proved by the results of genetic analysis, thereby contributing to cytogenetic characterisation.     

Determination of the correlation between stallion's age and number of sex chromosome aberrations in spermatozoa

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.     

Integrated immunohistochemical and DNA copy number profiling analysis provides insight into the molecular pathogenesis of canine follicular lymphoma

Follicular lymphomas (FLs) typically exhibit a chromosome translocation that induces constitutive expression of the anti‐apoptotic bcl2 protein and accumulation of additional molecular defects. This rearrangement offers a promising therapeutic target, but its nature as a fundamental driver of FL pathogenesis remains unclear as 15% of cases lack the translocation. We performed an integrated immunohistochemical and genomic investigation of 10 naturally occurring FL cases from domestic dogs, showing that, as with human tumours, they exhibit marked heterogeneity in the frequency and intensity of bcl2 protein expression. Genomic copy number aberrations were infrequent and broadly consistent with those of other canine B‐cell lymphoma subtypes. None of the canine FL specimens exhibited a rearrangement consistent with the hallmark translocation of human FL, despite their remarkable histomorphologic similarity. Parallel exploration of canine and human cases may reveal alternative tumour‐initiating mechanisms other than BCL2 disruption, yielding a more complete definition of the molecular pathogenesis of FL.     

60Co γ射线诱发谷稗和御谷的辐射生物学效应研究

研究利用60Co γ射线以10、20、30 Gy的剂量处理谷稗Echinochloa crusgalli和御谷Pennisetum glaucum 2种牧草种子,以未经处理的种子作为对照,进行了发芽、幼苗生长和根尖细胞染色体畸变的研究。结果表明,60Co γ射线辐照抑制了谷稗和御谷的种子萌发,不同程度刺激了幼苗的苗高增高。60Co γ射线辐照后,谷稗和御谷的根尖细胞中出现了单微核、双微核、单桥、双桥等各类的染色体畸变类型。随着60Co γ射线辐射剂量的增大,微核和染色体畸变率也逐渐增大,在30 Gy时达到最大。谷稗对于60Co γ射线辐射的敏感性要大于御谷。     

“实践八号”搭载8个苜蓿品种细胞学效应研究

通过“实践八号”育种卫星搭载8个苜蓿Medicago sativa品种干种子,研究苜蓿根尖细胞学效应。研究结果表明,航天诱变促进了8个苜蓿品种的根尖细胞有丝分裂活动,龙牧803有丝分裂指数的辐射生物损伤增加的幅度最大,草原1号增加的幅度最小。同时,航天诱变诱发了8个苜蓿品种根尖细胞产生染色体断片、染色体粘连、游离染色体、落后染色体等畸变类型,染色体断片是主要畸变类型,Pleven6总畸变率最大,肇东苜蓿总畸变率最小,初步推断8个苜蓿品种中Pleven6对航天诱变的敏感性最高,肇东苜蓿的敏感性最低。     

Molecular characterization of canine BCR‐ABL–positive chronic myelomonocytic leukemia before and after chemotherapy

Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High‐resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region‐Abelson murine leukemia viral oncogene homolog (BCR‐ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR‐ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.     

Short‐Term Antiandrogen Flutamide Treatment Causes Structural Alterations in Somatic Cells Associated with Premature Detachment of Spermatids in the Testis of Pubertal and Adult Guinea Pigs

In spite of widespread application of flutamide in the endocrine therapies of young and adult patients, the side effects of this antiandrogen on spermatogenesis and germ‐cell morphology remain unclear. This study evaluates the short‐term androgen blockage effect induced by the administration of flutamide to the testes of pubertal (30‐day old) and adult (65‐ and 135‐day old) guinea pigs, with an emphasis on ultrastructural alterations of main cell types. The testes removed after 10 days of treatment with either a non‐steroidal antiandrogen, flutamide (10 mg/kg of body weight) or a pharmacological vehicle alone were processed for histological, quantitative and ultrastructural analysis. In pubertal animals, flutamide androgenic blockage induces spermatogonial differentiation and accelerates testes maturation, causing degeneration and detachment of primary spermatocytes and round spermatids, which are subsequently found in great quantities in the epididymis caput. In post‐pubertal and adult guinea pigs, in addition to causing germ‐cell degeneration, especially in primary spermatocytes, and leading to the premature detachment of spherical spermatids, the antiandrogen treatment increased the relative volume of Leydig cells. In addition, ultrastructural evaluation indicated that irrespective of age antiandrogen treatment causes an increase in frequency of organelles involved with steroid hormone synthesis in the Leydig cells and a dramatic accumulation of myelin figures in their cytoplasm and, to a larger degree, in Sertoli cells. In conclusion, the transient exposition of the guinea pigs to flutamide, at all postnatal ages causes some degenerative lesions including severe premature detachment of spermatids and accumulation of myelin bodies in Leydig and Sertoli cells, compromising, at least temporarily, the spermatogenesis.     

卫星搭载对羊草种子萌发及细胞学效应的研究

试验研究了"实践八号"航天育种卫星搭载羊草种子后的萌发及根尖细胞学效应。结果表明:太空诱变后羊草种子的萌发能力降低,但与对照差异不显著(P>0.05);太空诱变促进了羊草根尖细胞的有丝分裂活动,分裂指数较对照增加24.17%;太空诱变使羊草根尖细胞在有丝分裂过程中产生微核、染色体断片、染色体粘连、落后染色体、游离染色体等畸变类型。其中,单微核是主要的核畸变类型,染色体断片为主要的染色体畸变类型。"实践八号"卫星搭载对羊草根尖细胞有明显的诱变效应。     

Structural abnormalities of the X-chromosome in a heifer.

Structural chromosome abnormalities, some of them unusual, were found in 18 (36%) of 50 photomicrographed mitotic cells of a blood lymphocyte culture from a three month old Holstein heifer affected with chronic tympanism of the rumen. The aberrations involved more specifically the X-chromosome. The etiology of the anomalies and the cause of their X-chromosome propension are of unknown nature.     

青海细毛羊,新疆细毛羊和藏系绵羊染色体比较研究

采用外周血微量全血培养法研究了生活在青海省三角城种羊场的青海细毛羊、新疆细毛羊、藏系绵羊的染色体核型及畸变。结果表明：三种绵羊二倍体细胞的染色体数目均为２ｎ＝５４，ＮＦ＝６２，公羊核型为５４，ＸＹ；母羊为５４，ＸＸ。染色体畸变类型主要为结构断裂和多倍体，其频率分别为：青海细毛羊１．５３０％和１．８４４％；新疆细毛羊２．２９２％和１．５６８％；藏系绵羊２．０２２％和１．１０３％，并对三种羊的畸变类型进行下检验，结果差异均为极显著（Ｐ＜０．０１）。     

杀菌剂多菌灵在桑叶中的消解动态研究

多菌灵(carbendazim)是家蚕微粒子病的防治药物。将50%多菌灵可湿性粉剂分别稀释至1000和500倍药液后喷洒桑树,用高效液相色谱(HPLC)检测桑叶中多菌灵的残留和消解动态。结果表明:桑树喷洒稀释1000倍的多菌灵药液后,桑叶中药物的消解动态方程为Ct=4.5339e-0.2215t,药物的半衰期T1/2=3.13d;喷洒稀释500倍的多菌灵药液后,桑叶中药物的消解动态方程为Ct=6.0527e-0.1030t,药物的半衰期T1/2=6.73d。多菌灵在桑叶中的残留与消解动态显示低浓度(稀释1000倍)的药液在桑叶中的降解更快,所以建议生产上适当采用高浓度药液喷施桑树,并根据相应的药物消解动态数据确定桑叶采摘时期,以保证家蚕食下药叶对微粒子病的防治效果。     

Determination of carbendazim residues in the eggs, liver and pectoral muscle of Japanese quail (Coturnix coturnix japonica)

The effect of carbendazim, a widely applied cereal seed dressing agent, was studied in Japanese quail (Coturnix coturnix japonica) during an overall period of eight weeks, consisting of a four-week feeding phase and a subsequent four-week excretion period. Body mass and feed consumption of the birds were monitored and residues of the active ingredient were determined by an analytical chemical method. During the eight-week study period, changes (either decrease or increase) directly attributable to the toxic effects of carbendazim were not found either in body mass or in feed consumption. Active ingredient levels exceeding the limit of detection were found in the liver (average: 0.0262 mg/kg) and pectoral muscle (average: 0.0236 mg/kg) of the birds and also in the egg (0.0338 mg/kg) samples. From the results it can be concluded that through the consumption of cereal seeds dressed with carbendazim, this active ingredient can be incorporated into the tissues of animals. Via the food chain, carbendazim can also enter the human organism, where it may cause various pathological changes in interaction with other chemicals.     

两种防霉剂与丙酸的组合对青贮牧草根霉菌毒力的研究

采用抑菌圈法研究了75%百菌清、50%多菌灵与99.5%丙酸在不同浓度下组合对高寒地区青贮饲草根霉菌的毒力。结果表明:丙酸(200mg/kg) 百菌清(400mg/kg)、丙酸(200mg/kg) 多酸灵(300mg/kg)、丙酸(100mg/kg) 多菌灵(300mg/kg)组合对根霉菌的毒力没有显著差异,但其毒力均极显著地高于其他处理;以丙酸(200mg/kg) 百菌清(400mg/kg)组合对青贮牧草根霉菌的抑菌作用最佳,其毒力分别是丙酸(200mg/kg) 多菌灵(300mg/kg)组合的1.05倍和丙酸(100mg/kg) 多菌灵(300mg/kg) 组合的1.20倍。     

Microdissection of chromosomes in veterinary medicine

Microdissection of chromosomes and subsequent amplification of the DNA material by PCR allow the development of libraries, region-specific DNA probes or chromosome painting probes. Veterinary medicine will benefit from this method in instances where clarification of numercal or structural chromosome aberrations is needed.     

Analysis of complex cytogenetic alterations in three canine mammary sarcomas.

Cells of mammary sarcomas in three dogs were analysed cytogenetically. In an osteoidsarcoma, hyperdiploidy with a range of 92 to 98 chromosomes, and several structural aberrations (for example, a derivative chromosome 1, isochromosome 13 and several bi-armed markers) were observed. Isochromosome 13 was also present in a case of an osteoidchondrosarcoma. In a case of chondro-osteosarcoma both hypodiploidy with a chromosome range of 60 to 66 and hyperdiploidy with a range of 115 to 128 and several centric fusions were observed.     

Cytogenetic study of bovine leukosis

Nine dairy cows aged four to eleven years were subjected to examination by chromosomal analysis. These cows had been found, by haematological examination, to suffer from leucosis. The study also covered one ten-day-old calf - heifer. The test group included two dam-daughter pairs. The animals belonged to the Black-Pied Lowland breed. The blood was sampled from vena jugularis and the karyotypes were processed and evaluated by the method after Moorhead et al. (1960), modified by Lojda et al. (1974). A list was kept for each animal. The tested animals were included in classes by the percentages of the chromosome aberrations: class I - two animals (up to 10% of aberrations), class II - seven animals (from 10% to 20% of aberrations), class III - one animal (above 20% of aberrations). Hyposomy was found in all cases, polysomy and hyperploidy in four cases. Structural aberrations were observed in nine cases, breaks being the most frequent anomalies (7 cases). Breaks on sexual chromosomes were observed in five cases, including the dam-daughter pairs; centric fusion occurred in one case and mixed aberrations in two cases.     

Cytogenetic studies in peripheral blood of bovines afflicted by papillomatosis

Ten types of bovine papillomavirus (BPV) have been described and there are reports of viral transmission via blood. The presence of viral DNA in lymphocytes was described to be associated with chromosome instability in these cells. This study presents an evaluation of chromosome instability in short-term peripheral lymphocyte cultures from cows presenting skin papillomatosis, compared with asymptomatic infected animals and non-infected healthy bovines. In a total of 2203 cells, 918 (42%) showed at least one chromosome aberration: 42.7 (± 7.8) in animals with papillomatosis (BPV + W), 40.2 (± 11) in asymptomatic animals (BPV-W) and 4 (± 2) in control animals. Significant differences were found between the infected group (with or without symptoms) and the control group (P < 0.0001). The increased frequencies of chromosome aberrations suggest an interaction between the virus and host cell chromatin.     

多菌灵在果园白三叶青贮中的降解及其对微生物群落的影响

为了解白三叶青贮后不同浓度多菌灵农药的降解情况和果园喷施多菌灵对白三叶青贮过程中细菌多样性的影响，扩大具有农药残留的果园覆盖植物利用途径，在喷施不同浓度［2.0 （RU^-），2.5 （RU），3.0 g·L^-1 （RU⁺）］的多菌灵后进行白三叶青贮，以喷水为对照，发酵60 d。采用Miseq高通量测序技术，分别在青贮开始前、青贮第3和60天取样，对白三叶中细菌群落多样性进行研究。结果表明，青贮后多菌灵降解率达到59.6%以上，喷施量越高，降解率越高；多菌灵处理显著（P<0.05）增加了白三叶青贮发酵的乳酸、乙酸、丙酸含量，乳酸在多菌灵RU^-、RU、RU⁺浓度下分别增加了47.55%、63.24%、71.08%，乙酸分别增加了24.49%、44.90%、46.94%，丙酸分别增加了66.67%、187.50%、250.00%，有利于白三叶青贮饲料营养成分的保留；多菌灵喷施显著（P<0.05）改变了白三叶青贮菌群群落构成，增加了青贮的菌群丰度、多样性；多菌灵处理中乳球菌属、魏斯氏菌属、泛菌属、Rosenbergiella、假单胞菌属、寡养单胞菌属、肠杆菌属、芽孢杆菌属菌群丰度增加，不利于青贮发酵，但随着青贮时间的推移，寡养单胞菌属、肠杆菌属、芽孢杆菌属等菌群丰度减少。喷施多菌灵后有利于青贮发酵微生物菌群的丰度增加，对青贮发酵有害菌、多菌灵降解菌群丰度及青贮微生物多样性产生影响，青贮后多菌灵残留高于欧洲食品安全局规定的作物类动物饲料中农药最大残留量，达不到饲用标准。研究结果可为具有农药残留的白三叶青贮饲料的饲用安全性和果园覆盖植物资源的开发利用提供理论依据。     

Quercetin induces structural chromosomal aberrations and uncommon rearrangements in bovine cells transformed by the E7 protein of bovine papillomavirus type 4

Bovine papillomavirus type 4 (BPV‐4) and bracken fern are cofactors in the carcinogenesis of the upper gastrointestinal (GI) tract of cattle. An experimental in vitro model system has been developed to analyse the co‐operation between the viral transforming protein E7, the cellular ras oncogene and quercetin, one of the mutagens of bracken fern, during neoplastic progression of primary bovine cells. We now report cytogenetic studies of these cells at different stages of malignant transformation: parental primary non‐transformed PalF cells; E7R cells transformed by BPV‐4 E7 and activated ras but not tumorigenic, and tumorigenic E7Q cells derived from E7R cells after treatment with quercetin. All cell lines presented increased numbers of aneuploid cells. The rate of structural chromosomal aberrations observed was increased in transformed cells. In addition, E7Q cells showed chromosomes with peculiar rearrangements, which resulted in metacentric and submetacentric marker chromosomes, with an increase in the mean chromosome arm number. These markers were the products of possible centric fusions. These aberrations and rearrangements were distributed throughout the karyotype, no specific chromosome was involved and the heterochromatic centromeric regions appeared to be preserved.     

Teratogenic and cytogenetic effects of ivermectin and its interaction with P-glycoprotein inhibitor

Experiments in animals proved that P-glycoprotein (Pgp) forms a functional barrier between maternal and fetal blood circulation in the placenta, thus protecting the fetus from exposure to xenobiotics during pregnancy. In this study we aimed to demonstrate the effects of administration of ivermectin (anthelmentic drug, Pgp substrates), either alone or simultaneously with verapamil (Pgp inhibitor) in Wister rats on fetal development, maternal bone marrow for detection of micronuclei (MN), chromosomal aberrations and mitotic index (MI) and embryonic liver cells for cellular proliferation indicated by MI, and bleeding from umbilical vessels for detection of embryonic micronuclei (MN). The results revealed that administration of ivermectin or verapamil at 6th through 15th day of gestation did not significantly altered fetal development. While, co-administration of ivermectin and verapamil clearly disturbed fetal development as indicated from abnormal feto-maternal attachment and a significant decrease in fetal weights and numbers. Furthermore, co-administration of both drugs induced a significant increase in resorption sites, post-implantation loss and external, visceral and skeletal abnormalities. They also induced genotoxicity in both dam and embryonic cells indicated by reduced mitotic index, increased number of micronucleated erythrocytes in both, and increased different types of chromosomal aberrations in dam cells, while ivermectin alone show some genotoxic effect on somatic cells of dams and the embryos. Verapamil induced reduction of embryonic mitotic index. We concluded combined treatment of ivermectin and verapamil severely affect fetal genetic material and development and induced genotoxic effect in somatic cells of the dams.