A new molecular screening tool for the detection of chromosomal abnormalities in donkeys

Chromosomal abnormalities are a major cause of infertility and reproductive problems in equids. Nowadays, their detection is rising due to the use of new diagnostic tools based on molecular markers instead of karyotyping. Reports of this kind of genetic aberrations in domestic donkeys (Equus asinus) are extremely scarce, despite their importance in human activities. In the present study, we analysed the implementation of a short‐tandem‐repeat (STR)‐based molecular method initially developed for horses, as a diagnostic tool to detect chromosomal abnormalities in donkeys. The frequency of five X‐linked (LEX003, LEX026, TKY38, TKY270 and UCEDQ502) and one Y‐linked (ECAYM2) molecular markers and one Y‐linked gene (sex‐determining region Y, SRY) was characterized in 121 donkeys from two diverse breeds, the Spanish Andalusian and the African Moroccan breeds. The molecular panel showed 100% sensitivity and 99.67% specificity in detecting 10 different chromosomal abnormalities in the species. In conclusion, this methodology is a valid, rapid and low‐cost tool for the detection and characterization of chromosomal abnormalities in domestic donkeys.     

Intersexuality in horses

Intersexuality is a rare congenital anomaly of horses. Diagnosis of intersexuality is difficult because there are usually no specific changes in the reproductive tract visible. During a period of five years, ten patients with reduced fertility or suspected intersexuality respectively were investigated using cytogenetic, molecular genetic, histopathological and endocrinological methods. In one case a 64,XX/63,X0 mosaicism was found. In six cases male pseudohermaphroditism was verified. These patients showed a male karyotype, testes and rudimentary parts of a female reproductive tract were present. One horse was suspected to be a male pseudohermaphrodite but the gonads were not examined. One horse was suspected to be affected by an XX-sex several syndrome and in one case a SRY-negative XY-sex reversal syndrome was most likely. In the case of an XX-sex reversal syndrome, there is a female chromosomal constitution, an uterus and cranial parts of the vagina are present but also testes tissue and possibly an enlarged penis like clitoris. Here an XX-sex reversal syndrome was suspected but not confirmed as it was not possible to examine the gonads and verify tissue from testes. Therefore a pseudohermaphroditismus femininus could not be excluded. In cases of XY-sex reversal syndrome the patients show a male chromosomal constitution, parts of a female reproductive tract but no testes tissue is present. For the horse described here, a deletion of the SRY gene was the most likely cause for the XY-sex reversal syndrome.     

Genetics of upper and lower airway diseases in the horse

Genetic predispositions for guttural pouch tympany, recurrent laryngeal neuropathy and recurrent airway obstruction (RAO) are well documented. There is also evidence that exercise‐induced pulmonary haemorrhage and infectious diseases of the respiratory tract in horses have a genetic component. The clinical expression of equine respiratory diseases with a genetic basis results from complex interactions between the environment and the genetic make‐up of each individual horse. The genetic effects are likely to be due to variations in several genes, i.e. they are polygenic. It is therefore unlikely that single gene tests will be diagnostically useful in these disorders. Genetic profiling panels, combining several genetic factors with an assessment of environmental risk factors, may have greater value, but much work is still needed to uncover diagnostically useful genetic markers or even causative variants for equine respiratory diseases. Nonetheless, chromosomal regions associated with guttural pouch tympany, recurrent laryngeal neuropathy and RAO have been identified. The association of RAO with other hypersensitivities and with resistance to intestinal parasites requires further study. This review aims to provide an overview of the available data and current thoughts on the genetics of equine airway diseases.     

Type IV rectal prolapse secondary to a long‐standing urinary bladder lithiasis in a donkey

Rectal prolapse has been attributed to conditions generating tenesmus or increasing abdominal pressure in both horses and donkeys. This report presents the case of a 7‐year‐old gelded donkey that sustained a type IV rectal prolapse secondary to a long‐standing cystic calculus after several episodes of intermittent mucosal prolapse. Rectal prolapse was addressed first on an emergency basis by colorectostomy. After medical stabilisation, pain and straining management with repeated epidural drug administrations, a laparoscopic‐assisted cystotomy was performed one week later to remove the calculus. The donkey was discharged and resumed his normal life at pasture.     

驴辅助繁育技术研究进展

目前,驴产业正在由短期育肥向繁育兼顾的新型可持续发展模式转变,同其他畜禽品种类似,驴产业的转型也是以扩大优良种群和提高品种质量为前提基础,而数量的增加和质量的提高都离不开良种繁育这一关键环节。然而,母驴繁殖力低是制约驴产业成功转型的主要瓶颈之一。驴繁殖力低不仅导致现有驴群的扩繁速度慢,存栏量与日益增长的皮、肉、奶的消费需求之间差距越来越大,而且使新品种的培育周期延长。已有研究表明,辅助繁育技术在提高母驴繁殖性能和改良驴的遗传性状等方面发挥重要作用,如同期发情、排卵控制、人工授精和胚胎移植等辅助繁殖技术可大幅提高母驴的繁殖能力;同时,微卫星标记技术、线粒体DNA测序技术、单核苷酸多态性研究和全基因组选择技术等分子育种手段也能够进一步加快驴的遗传改良进程。文章简要介绍了驴的生殖生理,系统阐述了同期发情、排卵控制、人工授精、胚胎移植、分子标记辅助育种和全基因组选择等辅助繁育技术在驴上的研究进展,并对未来研究方向进行了展望,以期对提高母驴繁殖性能提供借鉴,为开展驴分子育种研究提供有效线索。     

广灵驴分子系谱的建立及群体遗传结构分析

旨在对广灵县优种驴场保种群体进行调查的基础上构建分子系谱,并对其种群的遗传结构进行分析。本研究采集保种群成年、体况良好的广灵驴（体重350~400 kg）颈静脉血10 mL（n=107）,其中公驴13份,母驴94份,抗凝处理后提取全血DNA。采用12个微卫星标记进行荧光PCR扩增后,用ABI3730测序仪进行分型。分型结果采用Cervus 2.0和Pedigraph 2.4软件构建分子系谱,同时采用STRUCTURE2.3和Fstat软件计算群体遗传参数,采用R语言的hclust函数绘制7头公驴及其后代的系统发生NJ树（邻接树）。结果,对107头种驴进行了分子系谱构建,找到了30头子代的父亲和7头子代的母亲,系谱可靠性>90%;微卫星标记的平均观测杂合度（H_Obs）和多态信息含量（PIC）分别为0.676 5和0.593 9,标记遗传多样性较高;NJ树对7个公驴家系进行了聚类;群体分化系数（F_ST）为0.184,群体平均近交系数（F_IT）为0.033,群体内近交系数（F_IS）为-0.238,且群体处于哈代-温伯格平衡状态,存在很弱的近交。本研究建立了广灵驴保种群可靠性较高的分子系谱并对其遗传结构进行了分析,证明该群体遗传多态性较高,群体近交系数较低,处于较好的保种状态,具有较大的品种资源开发潜力。     

Association of owner-reported noise with findings during dynamic respiratory endoscopy in Thoroughbred racehorses

Reasons for performing study: To determine the association between owner‐reported noise and findings during dynamic respiratory endoscopy (DRE) in a large case series. Hypothesis: The sensitivity of owner‐reported noise for dynamic upper respiratory tract obstructions in horses is low, and the specificity is high. Methods: One hundred horses underwent DRE for the investigation of abnormal respiratory noise and/or poor performance. The association of abnormal noise with findings during DRE was evaluated. Results: Eighty‐five horses underwent DRE for the investigation of abnormal respiratory noise. Of these, 82% were found to have one or more obstructive upper respiratory tract abnormalities during DRE. Forty‐eight percent of horses reported to gurgle, rattle or make a rough noise were diagnosed with solitary palatal dysfunction. A further 24% with this history showed palatal dysfunction in combination with an additional abnormality. Twenty‐seven percent of horses with a history of whistling or roaring showed some degree of recurrent laryngeal neuropathy. Seven percent of horses with a history of whistling or roaring had vocal cord collapse as a solitary condition, whereas 40% had vocal cord collapse and another abnormality. The sensitivity of abnormal respiratory noise for any obstruction of the upper portion of the respiratory tract was high (84%), while the specificity was low (25%). Characteristic owner reported noise patterns showed moderate to low sensitivity for specific conditions. Whistling and roaring showed the highest specificity (≥80%) for laryngeal dysfunction. Conclusion: Diagnosis of upper respiratory tract obstructions based solely on owner‐reported noise and performance history may result in incomplete diagnoses. Clinical relevance: DRE should be performed in horses with abnormal respiratory noise to rule out complex conditions of the upper portion of the respiratory tract.     

Surgical correction of urethral dilatation in an intersex goat.

Multiple congenital urethral abnormalities were successfully corrected in a polled goat kid. Anatomic genito-urinary abnormalities identified were paired testes with associated epididymis, ductus deferens, and active endometrial tissue. Blood karyotyping revealed the female state--XX sex chromosomes. This case exemplifies the complex interactions in addition to Y dominant Mendelian genetics that determine reproductive tract development in goats. The resultant intersex state is clinically recognized with greater frequency in polled progeny.     

Highly diverse type of equine arteritis virus (EAV) from the semen of a South African donkey: short communication

Equine arteritis virus (EAV) was detected by RT-nested PCR in semen samples from a naturally infected South African donkey. Sequence analysis of the amplified ORF5 fragment revealed only 60 to 70% nucleotide identity to a panel of EAV reference sequences. The unique donkey EAV sequence was also found to be stable during passage in horses. The sequence data reported in this study indicate that the South African donkey variant might represent a new genotype of EAV. The distinct genetic properties of the South African asinine strain of EAV suggest a divergent evolution of this arterivirus in various host species or, alternatively, a possible role for African donkeys in the emergence of EAV in horses.     

Biochemical and genetic evaluation of the role of AMP-activated protein kinase in polysaccharide storage myopathy in Quarter Horses

OBJECTIVE: To evaluate whether biochemical or genetic alterations in AMP-activated protein kinase (AMPK) play a role in the development of polysaccharide storage myopathy (PSSM) in Quarter Horses. ANIMALS: 30 PSSM-affected and 30 unaffected (control) Quarter Horses. PROCEDURES: By use of an established peptide phosphotransfer assay, basal and maximal AMPK activities were measured in muscle biopsy samples obtained from 6 PSSM-affected and 6 control horses. In 24 PSSM-affected and 24 control horses, microsatellite markers identified from the chromosomal locations of all 7 AMPK subunit genes were genotyped with a fluorescent DNA fragment analyzer. Alleles of 2 of the AMPK gamma subunit genes were genotyped via DNA sequencing. Allele frequencies of DNA markers in or near the AMPK subunit genes were measured in isolated genomic DNA. RESULTS: No differences in basal or maximal muscle AMPK enzyme activities between PSSM-affected and control horses were detected. There were also no differences in allele frequencies for microsatellite markers near any of the 7 AMPK subunit genes between the 2 groups. Furthermore, previously known and newly identified alleles of 2 equine AMPK gamma subunit genes were also not associated with PSSM. CONCLUSIONS AND CLINICAL RELEVANCE: These results have provided no evidence to indicate that AMPK plays a causative role in PSSM in American Quarter Horses.     

Prevalence of lungworm D. arnfieldi (Cobbold 1884) in donkeys in Denmark and in horses in herds together with donkeys (author's transl)

During a 5 months' period from January to May 1981 faecal samples of 176 donkeys from 59 herds all over the country were examined at the Institute of internal medicine, The Royal Veterinary and Agricultural University, Copenhagen. In addition, the investigation also included 106 horses from 30 of the donkey herds and 34 horses hospitalized with symptoms of dyspnoea and chronic coughing. In all, 87.5% of the donkeys were shown to excrete D. arnfieldi larvae, often in very high numbers, and the larval excretion was the only symptom of lungworm infection. There was no significant correlation between larval excretion and age, sex or herd size. In horses, the frequency of D. arnfieldi infection was approximately the same in the horses from donkey herds and in horses hospitalized due to chronic cough (10.4% and 8.8% resp.). In contrast to the donkeys, the infection in horses was always accompanied by dyspnoea and coughing.     

Karyotype evaluation among young horse populations in Poland

Five hundred young horses of the following breeds: Thoroughbred, Silesian, Malopolska, Wielkopolska, Polish Konik, Hutsul, Shetland Pony, Half-bred Anglo-Arabian, Noble Half-bred, Fjord and crosses were cytogenetically investigated. Chromosome preparations obtained after lymphocyte culture were analysed using conventional Giemsa staining and CBG-banding methods. In the case of abnormalities GTG-banding as well as FISH technique were applied. In ten mares different karyotypic abnormalities were diagnosed. One mare showed chromosome chimerism (64,XX/64,XY), eight had sex chromosomal aneuploidy (one in pure line 63,X and seven in mosaic form 63,X/64,XX) and one presented autosomal aneuploidy with mosaicism (64,XX/65,XX,+31). The influence of sex chromosome abnormalities on fertility and the possible utilisation of karyotypic control in any selection programme are discussed.     

Repeated Early Embryonic Loss in a Thoroughbred Mare with a Chromosomal Translocation [64,XX,t(2;13)]

Peripheral blood samples from a 13-year-old Thoroughbred mare were submitted for chromosome analysis. The mare had a poor reproduction record only producing four live foals during her 10 years as a broodmare. She had remained barren or experienced early embryonic loss in the other 6 years. Chromosomal analysis revealed the mare carried a rare nonreciprocal translocation involving chromosomes 2 and 13 [64,XX,t(2;13)]. Fluorescence in situ hybridization with probes specific for horse chromosomes 2 and 13 were used to confirm the nonreciprocal translocation. Both conventional and molecular cytogenetic techniques are important for identifying and characterizing chromosomal abnormalities in horses with poor reproductive performance, particularly in mares experiencing repeated early embryonic loss.     

Intervertebral disc disease of the cervical and cranial thoracic vertebrae in equidae: eight cases

Intervertebral disc disease in the cervical and cranial thoracic vertebrae is unusual in horses and the majority of documented cases have been associated with infection and resulted in ataxia. The current retrospective study documents the clinical and imaging features, and outcome in eight Equidae with neck stiffness ± forelimb lameness (n = 3) or ataxia (n = 2) assessed during a 10-year period at two clinics. The Equidae (one donkey and seven horses) ranged in age from 1.5 to 12 years (median 5.5 years). The duration of clinical signs ranged from 1 to 6 months (median 1.5 months). The donkey had a depressed demeanour. All Equidae had reduced range of neck movement. The donkey and one horse showed mild and severe ataxia respectively. Two horses showed a propensity to stumble on each forelimb, one of which exhibited forelimb lameness on the lunge or ridden. Two additional horses showed lameness in hand. One horse experienced ‘neck locking’ during grazing. Radiological abnormalities were identified involving the intervertebral symphysis between the sixth cervical vertebra (C6) and C7 in four Equidae; in two horses the articulation between C7 and the first thoracic vertebra (T1) was involved. One horse had abnormalities of the intervertebral symphyses of both C7 and T1, and T1 and T2. In one horse the articulation between C2 and 3 was affected. The donkey was treated with a prolonged course of doxycycline and improved. An advanced dressage horse returned to full-function after surgical fusion of the affected intervertebral symphysis. Intervertebral disc disease is a rare cause of neck stiffness ± lameness or ataxia.     

Fertility Assessment in Sorraia Stallions by Sperm‐Fish and Fkbp6 Genotyping

The Sorraia, a critically endangered indigenous Iberian horse breed, is characterized by low genetic variability, high rate of inbreeding, bad sperm quality and subfertility. Here, we studied 11 phenotypically normal but subfertile Sorraia stallions by karyotyping, sex chromosome sperm‐FISH and molecular analysis of FKBP6 – a susceptibility locus for impaired acrosome reaction (IAR). The stallions had normal sperm concentration (>300 million cells/ml), but the numbers of progressively motile sperm (21%) and morphologically normal sperm (28%) were invariably low. All stallions had a normal 64,XY karyotype. The majority of sperm (89%) had normal haploid sex chromosome content, although 11% of sperm carried various sex chromosome aneuploidies. No correlation was found between the percentage of sperm sex chromosome abnormalities and inbreeding, sperm morphology or stallion age. Direct sequencing of FKBP6 exon 4 for SNPs g.11040315G>A and g.11040379C>A revealed that none of the stallions had the susceptibility genotype (A/A‐A/A) for IAR. Instead, all animals had a G/G‐A/A genotype – a testimony of low genetic variability. The findings ruled out chromosomal abnormalities and genetic predisposition for IAR as contributing factors for subfertility. However, low fertility of the Sorraia stallions could be partly attributed to relatively higher rate of sex chromosome aneuploidies in the sperm.     

Analysis of pedigree and conformation data to explain genetic variability of the horse breed Franches-Montagnes

Franches‐Montagnes is the only native horse breed in Switzerland, therefore special efforts should be made for ensuring its survival. The objectives of this study were to characterize the structure of this population as well as genetic variability with pedigree data, conformation traits and molecular markers. Studies were focused to clarify if this population is composed of a heavy‐ and a light‐type subpopulation. Extended pedigree records of 3‐year‐old stallions (n = 68) and mares (n = 108) were available. Evaluations of body conformation traits as well as pedigree data and molecular markers did not support the two‐subpopulation hypothesis. The generation interval ranged from 7.8 to 9.3 years. The complete generation equivalent was high (>12). The number of effective ancestors varied between 18.9 and 20.1, whereof 50% of the genetic variability was attributed to seven of them. Genetic contribution of Warmblood horses ranged from 36% to 42% and that of Coldblood horses from 4% to 6%. The average inbreeding coefficient reached 6%. Inbreeding effective population size was 114.5 when the average increase of the inbreeding coefficient per year since 1910 was taken. Our results suggest that bottleneck situations occurred because of selection of a small number of sire lines. Promotion of planned matings between parents that are less related is recommended in order to avoid a reduction of the genetic diversity.     

Pedigree information reveals moderate to high levels of inbreeding and a weak population structure in the endangered Catalonian donkey breed

The Catalonian donkey is one of the most endangered donkey breeds in the world. At present, five main subpopulations exist: AFRAC, which consists of many genetically connected Catalonian localities; Berga, which consists of a single herd located also in Catalunya but under private management; and three minor non‐Catalonian subpopulations (Huesca, Sevilla and Toledo). In this study, we analysed the pedigree information of the Catalonian donkey herdbook to assess the genetic diversity and population structure of the breed. We found that the Catalonian donkey has suffered an important loss of genetic diversity and moderate to high increases of inbreeding because of the abuse of a few individuals in matings. This scenario is mainly characterized by the fact that both the effective number of founders and ancestors for the whole population was 70.6 and 27, respectively, while the equivalent number of founders was 146.5 and the number of ancestors explaining overall genetic variability was 93. In addition, only 14% of animals born between the 1960s and 1970s were significantly represented in the pedigree. Our results also show that subpopulations where breeders exchanged reproductive individuals had low levels of inbreeding and average relatedness. One subpopulation, Berga, was reproductively isolated and showed high levels of inbreeding (F = 7.22%), with average relatedness (AR = 6.61%) playing an important role in increasing the values of these coefficients in the whole pedigree. Using genealogical F‐statistics we have found little evidence of population structuring (F_ST = 0.0083) with major genetic differences among non‐Catalonian subpopulations.     

Genetic conservation of five endangered Spanish donkey breeds

The genetic diversity of most livestock species is being reduced, and it is not possible to preserve all of these livestock breeds. In order to preserve as much of the genetic diversity as possible, we must first have a robust method of measuring genetic diversity among breeds. Three different methods of study that graphically represent relationships among breeds are presented; Weitzman's method, principal component analysis (PCA) and a neighbour‐joining tree with allele sharing. Diversity was evaluated on the basis of 15 microsatellite markers typed over a total of 513 DNA samples collected from five Spanish donkey breeds. Breed differentiation was confirmed by the clustering based on the genetic distances between individuals, which essentially grouped all individuals in discrete clusters. The genetic distance among breeds was used to measure the global diversity of the set in breeds considered, and to evaluate the marginal loss of diversity attached to each breed. The Catalana breed appeared to be the most ‘unique’ in the set considered. In addition to this, the usefulness of global evaluations of diversity using molecular markers to choose breeds is worthy of conservation.