Preliminary studies of a platelet function disorder in Simmental cattle.

A bleeding disorder due to abnormal platelet function occurs in Simmental cattle. Whole blood from these animals underwent good clot retraction. Platelet aggregation in response to adenosine diphosphate (ADP) and collagen in a whole blood aggregation system was markedly impaired. Normal bovine platelets in a whole blood aggregation system showed very little aggregation in response to epinephrine and arachidonic acid. Aggregation in platelet-rich plasma was negligible in response to ADP, collagen and thrombin. Dense granule release of radiolabelled serotonin from the platelets of one affected cow was similar to that of normal bovine platelets. Platelet membrane glycoprotein electrophoresis with the platelets of one affected cow revealed no quantitative abnormalities. These findings reveal similarities and differences in thrombopathic Simmental platelet function when compared to human Glanzmann's thrombasthenia and Basset Hound thrombopathia.     

Basset Hound thrombopathia in a 4‐month‐old female Ba‐Shar (Sharp Asset)

A 3‐month‐old female Basset Hound‐Shar Pei mix puppy (Ba‐Shar or Sharp Asset) presented with oral bleeding due to a cracked molar. On physical exam, an aural hematoma was also noted that the owner indicated was chronic. The puppy was hospitalized for over 24 h until the bleeding was brought under control. At 4 months of age, the puppy again presented with oral bleeding due to loss of deciduous teeth and was hospitalized until bleeding was controlled. Coagulation screening tests, platelet numbers, and von Willebrand Factor antigen levels were within reference limits. Based on the presence of platelet‐type bleeding in the face of normal screening test results, samples were submitted for DNA testing for Basset Hound thrombopathia. The puppy tested as affected for the calcium and diacylglycerol regulated guanine nucleotide exchange factor I (CalDAG‐GEFI) mutation causing this disorder. This is the first time thrombopathia has been diagnosed in a “designer” breed.     

Molecular and genetic basis for thrombasthenic thrombopathia in otterhounds.

OBJECTIVES: To determine the molecular and genetic basis for thrombasthenic thrombopathia in Otterhounds and establish whether the defect would be best classified as type-I Glanzmann's thrombasthenia. ANIMALS: 57 dogs, including 13 affected Otterhounds, 23 carrier Otterhounds, 17 unaffected Otterhounds, and 4 clinically normal unrelated dogs of other breeds. PROCEDURE: Functional (platelet aggregation, clot retraction, buccal mucosa bleeding time) and biochemical (electrophoresis, flow cytometry, fibrinogen content) analyses were conducted. In addition, first-strand cDNA synthesis from platelet total RNA was performed. Exons of the genes encoding for glycoproteins (GP) IIb and IIIa were amplified in overlapping fashion. The resulting products were excised from agarose gels and sequenced. The sequences obtained were compared with known cDNA sequences for canine GPIIb and GPIIIa. RESULTS: A single nucleotide change at position G1193 (1100) was detected in exon 12 of the gene encoding for platelet GPIIb in 2 affected Otterhounds. Carrier Otterhounds were heterozygous at this position, and 2 unaffected Otterhounds were unchanged. This nucleotide change would result in substitution of histidine for aspartic acid at position 398 (367) within the third calcium-binding domain of GPIIb. CONCLUSIONS AND CLINICAL RELEVANCE: These studies suggest that thrombasthenic thrombopathia of Otterhounds is homologous phenotypically and has a similar molecular basis to type-I Glanzmann's thrombasthenia in humans.     

Identification of an Intrinsic Platelet Function Defect in Spitz Dogs

A recently identified intrinsic platelet function defect in 2 Spitz dogs is described. Both affected dogs had a history of chronic intermittent bleeding primarily from the nasal, oral, and gastrointestinal mucosa. Platelet aggregation in response to adenosine diphosphate (ADP), collagen, and platelet activating factor (PAF) was absent; however, platelet shape change did occur. Platelets aggregated in response to gamma thrombin, although a delayed onset and a reduced velocity of aggregation were present. Platelet ¹⁴C-serotonin release was diminished in response to collagen and PAF. Glycoprotein Illa was detected on the surface of platelets by flow cytometry. Platelets were morphologically normal under light and electron microscopy. Two male Spitz dogs, related to one of the affected dogs, did not have a bleeding diathesis. Collagen-induced platelet aggregation, however, was diminished in these 2 dogs. This platelet defect most closely resembles the defect described in Basset hounds.     

牛肌肉生长抑制素基因单核苷酸多态性分析

应用PCR-SSCP分析方法,对94头肉牛(公牛45头:西门塔尔34头,夏洛来11头;母牛49头:西门塔尔24头,夏洛来25头)的肌肉生长抑制素(MSTN)3个外显子进行了多态性分析.结果显示,第1外显子存在2种基因型,分别为AA型和AB型.经测序发现,第1外显子4 bp处存在C→G的碱基突变,导致编码的氨基酸由谷氨酰胺(Gln)→谷氨酸(Glu).统计结果表明,等位基因B的含量低,而且只在西门塔尔品种内含0.026 6.利用SPSS软件作最小二乘分析,结果表明,B等位基因与西门塔尔牛的成年体质量和犊牛出生体质量呈显著正相关.     

Prophylactic use of a lyophilized platelet product for rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a dog with a P2Y12 platelet receptor mutation

Objective

To describe the periprocedural use of a lyophilized platelet product during rhinoscopic diagnosis and treatment of sinonasal aspergillosis in a Greater Swiss Mountain Dog with a P2Y12 platelet receptor disorder.

Case Summary

After the development of severe epistaxis, a Greater Swiss Mountain Dog was diagnosed with thrombopathia secondary to a P2Y12 receptor gene mutation. Concurrent primary nasal disease was also suspected due to persistent mucopurulent nasal discharge. One month after the initial presentation for epistaxis, the dog was readmitted for workup of nasal disease. Computed tomography of the head showed turbinate lysis and regional lymphadenopathy. Because of concern for a high risk of bleeding in a thrombopathic patient subjected to rhinoscopy and nasal biopsies, a lyophilized platelet product was administered prior to the procedure. Rhinoscopic exam revealed fungal plaques consistent with Aspergillus spp. that were later confirmed on fungal culture to be Aspergillus fumigatus. Rhinoscopic biopsies were performed as well as debridement of the fungal plaques, followed by topical administration of clotrimazole solution. Bleeding was minimal during and after the procedure, and the dog recovered uneventfully.

New or Unique Information Provided

This is the first report of the prophylactic use of lyophilized platelets in a thrombopathic patient undergoing an invasive procedure with potential for significant hemorrhage. Minimal bleeding occurred during the procedure, suggesting that lyophilized platelets could be used for the prevention of bleeding in thrombopathic patients undergoing invasive procedures.     

Correlations Analysis between Polymorphism of 3’-UTR in MSTN Gene and Slaughter Traits of China Red Steppe

In order to study the effect of myostatin (MSTN) gene on the meat performance of Local Yellow cattle in China,selecting 2 Local cattle breeds (Grassland Red bull,Yanbian cattle) as the research object,four binary hybrid cattle group (Germany Yellow cattle×Simmental,Limousin×Simmental,Charolais×Simmental and Red Angus×Simmental) as reference.PCR-RFLP and DNA sequencing methods were conducted to detect single nucleotide polymorphism of MSTN gene in two Chinese native cattle and four cross cattle,and analyzed the correlations between single nucleotide polymorphism of China Red Steppe MSTN gene and slaughter traits.The sequence analysis showed that there was no mutations in the MSTN gene exon 3,a C5358A mutation of 3'-UTR of MSTN gene was found,which made the AclⅠrestriction enzyme site disappeared,all cattle groups showed polymorphism which was formed two genes as A,C, and 3 genotypes as AA,CA and CC, respectively.The research results suggested that AA genotype in groups of four hybrid cattle had dominant advantage and the genotype frequency was apparently higher than two groups of Chinese Native cattle for the test of independence,allele A was expressed the preferential allelic,the frequencies of CA,CC and AA genotypes showed a little variation between two cross cattle and Native cattle,and the frequency of allele C was apparently higher than allele A,so allele C was designed the preferential allelic in the local population.While correlations between different genotypes of China Red Steppe and slaughter traits analysis showed that AA genotype individual net meat percentage and eye muscle area were significantly higher than CA and CC genotype individuals (P<0.05),but other slaughter straits had no significant differences in three genotypes(P>0.05).These results provided a useful reference for research of the meat performance of China Red Steppe in the molecular breeding field.     

Coat-colour dilution and hypotrichosis in Hereford crossbred calves

AIM: To investigate cases of coat-colour dilution and hypotrichosis in a group of Hereford x Friesian crossbred calves, and to define the underlying molecular genetics of the disorder. METHODS: The investigation was predicated on the hypothesis that this disorder was similar to a known dominantly inherited disorder of calves of black breeds crossed with Simmental cattle, for which there were candidate gene mutations. Sequence analyses of PCR amplicons from exon 1 and exon 11 of the premelanosome protein 17 gene (PMel17) were carried out. Restriction enzyme digestions of amplicons were followed using electrophoresis of digested fragments. RESULTS: It was shown that an affected calf and its Hereford sire were heterozygous for a three-base deletion in exon 1 of the PMel17 gene. These two animals were also heterozygous for a second mutation in exon 11 of the PMel17 gene. Four other related animals were likewise heterozygous for both mutations in the sire's herd of origin. CONCLUSIONS: Coat-colour dilution and hypotrichosis in Hereford crossbred calves in New Zealand is the same genetic disorder as that previously described in Simmental crossbred calves, and is linked to mutations in the PMel17 gene.     

A study of the inheritance of a bleeding disorder in Simmental cattle

A study was designed to determine the inheritance pattern of a blood platelet aggregation disorder in Simmental cattle utilizing embryo transfer technology. A Simmental donor cow that had previously produced a calf with the platelet aggregation disorder was superovulated and mated to a bull that had also produced affected offspring. Twenty-seven calves were produced from the 63 (42.9%) embryos transferred. This somewhat lower than expected pregnancy rate is suggestive of an increased rate of embryo loss. Twenty-three of 25 (92%) calves had normal platelet aggregation patterns and 2 failed to show any evidence of platelet aggregation. Data are suggestive that inheritance is not simple Mendelian recessive. A more likely scenario is that the defect is the result of the inheritance of at least 2 genes, which is also consistent with the sporadic incidence reported in the population at large.     

育成荷斯坦奶公牛与其他3个品种牛的肉品质比较研究

通过对育成荷斯坦奶公牛与西门塔尔牛、新疆褐牛及新疆土种牛肉品质部分指标的比较分析研究,旨在探讨荷斯坦奶公牛的肉品质。选择在相同营养模式下18月龄左右4个品种牛各3头进行屠宰,取右半边胴体的背最长肌作为肉品质试验样品,分别对牛肉的肉品质、常规营养成分及氨基酸含量进行测定和分析。结果表明,荷斯坦奶公牛肉色、失水率、系水力、熟肉率、大理石花纹等指标均优于新疆褐牛、新疆土种牛,次于西门塔尔牛;荷斯坦奶公牛嫩度优于新疆土种牛;粗蛋白质、粗灰分含量分别为20.14%、1.11%,且各品种间差异均不显著(P>0.05),干物质含量为26.30%,显著高于新疆褐牛和新疆土种牛(P<0.05),低于西门塔尔牛(P>0.05),粗脂肪含量为10.04%,显著高于其他品种牛(P<0.05);荷斯坦奶公牛含有人体需要的各种氨基酸,其中蛋氨酸、谷氨酸、甘氨酸、组氨酸、牛磺酸等含量丰富,氨基酸组成比例良好。     

Exclusion of NEU1 and PPGB from candidate genes for a lysosomal storage disease in Japanese Black cattle

A case of lysosomal storage disease has been reported in a calf of Japanese Black cattle. Lysosomal storage diseases are hereditary diseases caused by deficiency of lysosomal hydrolases. The clinical and pathological features and accumulated substrates of the affected animal indicated a possibility of sialidosis or galactosialidosis caused by deficiency of neuraminidase (NEU1) or protective protein for β-galactosidase (PPGB). In the present study, we investigated nucleotide sequences of the genes encoding these two proteins to evaluate whether mutation of these genes is involved in this disease. We determined cattle genomic sequences of these two genes by using bovine EST sequences and the nucleotide sequences of all exons of these genes were compared between affected and normal animals. The results showed several nucleotide substitutions, but none of them was a functional mutation or specific to the affected animal. Furthermore, genotyping of the microsatellite markers in the vicinity of these two genes revealed no homozygosity of the chromosomal regions including these genes in the affected animal. These findings indicated that neither NEU1 nor PPGB gene is responsible for the lysosomal storage disease of Japanese Black cattle and therefore the disease is neither sialidosis nor galactosialidosis.     

Clinical and laboratory findings of a bleeding disorder in Simmental cattle

Eight Simmental cattle were presented to the Western College of Veterinary Medicine over the past decade with historic or current prolonged episodes of bleeding. Spontaneous epistaxis, superficial hematomas, and prolonged bleeding following injury or minor surgery (such as tattooing) were frequent observations. Animals presented because of prolonged hemorrhage were anemic and debilitated. Compensatory erythropoiesis was compromised in these animals by depletion of iron stores due to chronic blood loss. Screening tests to assess coagulation were normal as were numbers and morphology of platelets. In vitro platelet aggregation was abnormal in all affected cattle. Three animals were euthanized due to uncontrollable hemorrhage but the precise source of hemorrhage was undetectable. These findings are consistent with bleeding due to a platelet function disorder which has been described previously in Simmental cattle.     

德系西门塔尔牛与荷斯坦牛及其杂种后代血液生理生化指标的比较分析

为探究德系西门塔尔牛、荷斯坦牛及其杂交后代血液生理生化指标状况及差异,选择5月龄、体重150~200 kg的健康公牛15头,其中荷斯坦公牛、西×荷杂种一代公牛(简称西荷杂种牛)、德系西门塔尔公牛各5头,育肥372 d后屠宰测定其各项血液生理生化指标。结果显示:德系西门塔尔牛红细胞、血红蛋白、红细胞压积均显著高于其他两组牛(P<0.01),白细胞、中性细胞数、中性细胞比率、嗜碱性粒细胞比率处于居中水平;荷斯坦牛白细胞、红细胞平均体积均显著高于其他两组牛(P<0.05),中性细胞数、中性细胞比率极显著高于其他两组牛(P<0.01),嗜碱性粒细胞比率则极显著低于其他两组牛(P<0.01);西荷杂种牛白细胞、游离甲状腺素、皮质醇均显著低于其他两组牛(P<0.05),淋巴细胞数、淋巴细胞比率、嗜酸性粒细胞比率、嗜碱性粒细胞数、嗜碱性粒细胞比率均极显著高于其他两组牛(P<0.01),血小板压积显著高于其他两组牛(P<0.05)。在血液生化指标方面,荷斯坦牛血清钾离子显著低于其他两组牛(P<0.05),德系西门塔尔牛游离甲状腺素浓度显著高于其他两组牛(P<0.05),荷斯坦牛皮质醇浓度显著高于其他两组牛(P<0.05),其他血液生理生化指标在3组间比较差异均不显著(P>0.05)。综上说明,西荷杂种牛抗病力和抗逆性较强,德系西门塔尔牛代谢旺盛,环境适应性强。     

Variation in the coding region of the prion protein gene in Slovak cattle

This study was conducted to investigate the presence of single nucleotide polymorphisms (SNPs) in the coding region of the bovine prion protein (PrP) gene among healthy and bovine spongiform encephalopathy (BSE-) affected cattle in Slovakia. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) followed by DNA sequencing were used to identify SNPs and variations in octapeptide repeats. Altogether three single nucleotide polymorphisms (g234a, c339t and c576t) and variations in the number of octapeptide repeat units (5 or 6) were found in the analysed part of the prion protein gene. All single nucleotide polymorphisms were silent, causing no amino acid changes. Significant differences (P < 0.05) in the genotype distribution of g234a polymorphism were observed when the homozygous genotype with a mutated allele (caa/caa) was compared to the heterozygous genotype -/cag among healthy and BSE-affected cattle. The homozygous genotype caa/caa was characteristic of the group of BSE-affected cattle. Additionally, the homozygous genotype caa/caa was significant for the group of Simmental crossbreeds among healthy cattle. The allele and genotype distribution of the other polymorphisms was not significantly different among groups of healthy and BSE-affected cattle. The possible influence of a silent mutation on expression of the gene is not clearly determined and needs further investigations.     

An inherited platelet function defect in a Simmental crossbred herd.

An inherited bleeding disorder, resembling Simmental hereditary thrombopathy (SHT), has been identified in a Simmental crossbred herd. In an affected bull calf, initially evaluated because of excessive bleeding from a vaccination site, the platelet aggregation response to the agonist, adenosine-diphosphate (ADP) was essentially absent and the aggregation response to platelet activating factor (PAF16) was reduced by at least 70%. The initial laboratory assessment of platelet function in the dam and sire yielded results which were within normal limits. The sire was not available for further testing. The dam, also a daughter of this sire, was subsequently shown to have a partially reduced aggregation response to ADP. Of 18 other offspring of the sire evaluated, 6 were also identified as having a partially impaired aggregation response. The maximum aggregation response to ADP and PAF16 in these 6 calves was approximately 50% of the level exhibited by unaffected animals. In contrast, the coagulation profiles were normal for all animals except for a heifer calf which also exhibited a partially impaired aggregation response. The plasma level of the coagulation protein, factor XI, was reduced in this heifer calf which suffered a fatal hemorrhage following dehorning. This report appears to be the 1st to have identified animals putatively heterozygous for SHT on the basis of the in vitro platelet aggregation response to ADP.     

甘肃金昌西门塔尔牛MSTN基因外显子多态性研究

[目的]为分析金昌地区西门塔尔牛 MSTN 基因三个外显子的遗传多态性及变异特征。[方法]采用 PCR-SSCP 方法检测了61头西门塔尔牛 MSTN 基因三个外显子的多态性。[结果]显示：金昌西门塔尔牛 MSTN 基因的第1外显子存在 A、B 两个等位基因以及AA、AB 两种基因型,其基因型频率分别为0.7705和0.2295;第2外显子存在 A、B 两个等位基因以及 AA、AB、BB 三种基因型,其基因型频率分别为0.0328、0.3443和0.6229;第3外显子只有 AA 一种基因型。序列分析表明,金昌地区的西门塔尔牛 MSTN 基因第1外显子在269bp 发生了 A→G 的突变和第2外显子在41bp 发生了 C→T 的突变,但都未导致氨基酸发生改变,属于同义突变;外显子3并未检测到突变。[结论]统计结果表明,金昌地区的西门塔尔牛 MSTN 基因第1外显子呈低度多态,第2外显子呈中度多态。     

甘肃武威西门塔尔牛MSTN基因外显子遗传变异的研究

为分析肉牛MSTN基因的遗传多态性及变异特征,采用PCR-SSCP方法检测了60头甘肃武威西门塔尔牛MSTN基因的多态性,对群体内各等位基因进行了克隆测序。结果显示：武威西门塔尔牛MSTN基因第1外显子存在A、B两个等位基因以及AA、AB两种基因型,基因型频率分别为0.9167、0.0833;第2外显子存在A、B两个等位基因以及AA、BB、AB三种基因型,基因型频率分别为0.5、0.25、0.25。序列分析表明,武威西门塔尔牛MSTN第1外显子在269 bp发生了A→G的突变,第2外显子在41 bp发生了C→T的突变,二者均属于同义突变。统计结果表明,武威西门塔尔牛MSTN基因第1外显子呈低度多态,第2外显子呈中度多态,外显子3没有发生突变。     

Nucleotide sequence of myostatin gene and its developmental expression in skeletal muscles of Japanese Black cattle

Myostatin, a member of the transforming growth factor‐β superfamily, is a well known negative regulator of skeletal muscle growth. In the present study, the 6660 bp nucleotide sequence of the myostatin gene in Japanese Black cattle (JBC), including the entire coding region of 1128 bp, was determined. The amino acid sequence deduced from the nucleotide sequence of JBC was well conserved with its sequence of other cattle, although it was found that an Α→G transition at nucleotide position 641 results in the substitution of asparagine by serine at amino acid position 214. In order to examine the expression pattern of the myostatin gene in the skeletal muscles of JBC, its expression in three skeletal muscles, Semitendinosus (ST) muscle, Biceps femoris muscle and Longissimus lumborum muscle, of fetal and calf stages was analyzed by real time polymerase chain reaction. The highest level of the myostatin expression was observed in the fetal stage. In calf stages the highest expression was observed in ST muscle compared with the other two muscles. These results suggest that a higher expression of myostatin gene, especially in the fetal stage and in ST muscle during calf stages, is involved in the arrest in skeletal muscle growth and that its functional domains and genomic structure in JBC are well conserved with those in other mammals.     

Genetic variation in monoamine oxidase A and analysis of association with behaviour traits in beef cattle

Husbandry of beef cattle requires animals that do not behave aggressively or timidly. The enzyme monoamine oxidase A and the coding gene (MAOA) play an important role in the complex regulation of behaviour. The complete coding region and a part of the non‐coding sequence of the bovine MAOA gene have been analysed in 20 German Angus and 20 German Simmental bulls and cows with the aim of detecting genetic variability. These two cattle breeds are known to differ regarding their behaviour during handling. Five single nucleotide polymorphisms (SNPs) were identified, three of which were found in the coding region of the gene (exons III and XV). One of the SNPs located in exon XV ( NC_007331.3 :g.80340C>T) was found to be a non‐synonymous mutation. The minor allele frequency of this resulting amino acid substitution was significantly different between 543 German Angus and 417 German Simmental calves (0.39 and 0.49, respectively). The potential functional impact of this polymorphism has been tested by in silico analysis, as well as by association analysis using behaviour scores of the genotyped calves for three behaviour tests that assessed the animals’ temperament during tethering, weighing or social separation. In silico analysis did not deliver consistent results arguing for or against a functional impact of the studied amino acid substitution on the function of the biological protein. No significant association was found between this MAOA polymorphism and the behaviour‐related scores analysed in the study.     

Effect of Temperature on Bovine Sperm Associated Antigen 11D Gene

To explore the influence of heat stress on the expression level of bovine sperm associated antigen 11D (SPAG11D),which was sperm membrane protein,the mRNA and protein expression levels of SPAG11D were tested in primary bovine testicular cells and sperm. The normal sperm and low activity sperm were collected from Jersey cattle, Bos bubalus and Simmental cattle. The results showed that mRNA and protein expression levels of SPAG11D were highest at 36℃ than others (32, 34, 38 and 40℃) after primary bovine testicular cells cultured for 24 h. And the mRNA and protein expression levels were higher in high temperature groups than low temperature groups. The mRNA expression of SPAG11D in 48 h was consistent with 24 h, but the protein expression levels decreased at high temperature (38 and 40℃). In addition, the protein expression of SPAG11D was lower in low activity sperm than in normal sperm (Jersey cattle and Bos bubalus), and there was no significant change in Simmental cattle sperm.