天津地区荷斯坦公牛CVM、DUMPS和CN遗传缺陷检测

本文旨在研究天津地区中国荷斯坦公牛脊椎畸形综合征（Complex vertebral malformation,CVM）、尿苷酸合酶缺乏症（Deftciencv of uridine monophospham synchase,DUMPS）和瓜氨酸血症（Citrullinemia,CN）3种遗传缺陷的携带者比率及系谱来源。通过PIRA—PCR和PCR—RFLP方法分别对天津奶牛发展中心参加全国青年公牛联合后裔测定和国家良种补贴项目的110头荷斯坦公牛进行了CVM、DUMPS和CN三种遗传缺陷检测。共发现6头CVM隐性有害基因携带公牛,携带者比例为5．45％,隐性有害等位基因频率为2．72％。经过系谱分析,其中4头CVM携带者均为美国公牛Carlin—MIvanhoeBell的后代,另外2头因系谱不完整而无法查询。未检测到DuMPs和CN隐性有害基因携带者。基于此,我国有必要尽快建立荷斯坦牛隐性遗传缺陷监控体系并进行系谱标注,避免携带公牛进入后裔测定和良种补贴项目,以逐步降低我国奶牛群体中遗传缺陷隐性等位基因频率。     

Identification of complex vertebral malformation carriers in Chinese Holstein.

Complex vertebral malformation (CVM) is a monogenic autosomal recessive hereditary defect of Holstein dairy cattle. It is caused by a point mutation from G to T at the nucleotide position 559 in bovine solute carrier family 35, member 3 gene (SLC35A3), which changes the amino acid sequence of uridine 5'-diphosphate-N-acetylglucosamine transporter protein from a valine to a phenylalanine in position 180. The elite U.S. Holstein sire Penstate Ivanhoe Star was identified as the common ancestor of the current CVM carriers. Because his offspring, mainly those of Carlin-M Ivanhoe Bell, were used in many countries, CVM has potentially spread into China. In the present study, using the polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) technique, 10 CVM carriers were found among 68 at-risk Chinese Holstein bulls, and 282 carriers were found among 602 at-risk cows. The results of this study indicate that the CVM gene exists in the Chinese Holstein population.     

Comparison of milk production of the progeny of BLAD-carrier and healthy Holstein bulls in Hungary.

'Bovine Leukocyte Adhesion Deficiency' (BLAD) is a recessive monofactorial, lethal inheritable defect occurring in Holstein-Friesian cattle and often passed on by well-known top bulls. The aim of this study was to find a relationship between the BLAD genotype of bulls, their genetic evaluation for milk and their daughters' milk production. BLAD-carrier and healthy bulls were compared on the basis of their breeding value published in November 1997. The first 100 bulls ranked according to the Total Production Index (TPI) were used, including nine BLAD carriers with 2,835 daughters and 77 healthy sires with 21,950 female progenies. For 14 bulls the BLAD genotype was not indicated. The healthy animals significantly outperformed the BLAD carriers, which result contradicts our earlier findings (Dohy et al., 1996; Jánosa and Dohy, 1997). In a BLAD elimination programme, the identification of BLAD carriers and properly planned mating are of great importance in order to avoid 'inter se' mating of BLAD-carrier top animals which can be of significant influence in Holstein breeding.     

Effects of a novel missense polymorphism within the SIGLEC5 gene on fertility traits in Holstein‐Friesian cattle

The aim of the study was to find functional polymorphism within two exons of the SIGLEC5 (sialic acid‐binding Ig‐like lectin‐5) gene and to examine its effects on the production and fertility traits of cows and bulls. Two hundred seventytwo Holstein‐Friesian cows and 574 bulls were included in the study. Novel missense polymorphism (A > G) within exon 3 causing substitution of amino acid arginine by glutamate in position 260 of SIGLEC5 protein (R260Q) was identified by sequencing and digestion by restriction enzyme Msp I. Basic production and fertility traits of cows and estimated breeding values (EBV) of bulls were analysed. The study demonstrated a significant association of SIGLEC5 R260Q polymorphism with days open and calving interval in cows as well as with breeding value for calving interval in bulls. An opposite effect of SIGLEC5 alleles for production and fertility traits was observed: the allele G increased the breeding value for the protein yield, while the allele A increased the breeding value for the calving interval. The current study suggests the involvement of SIGLEC5 R260Q polymorphism in biological processes related to fertility traits. This finding can be applied as a biomarker for a genetic improvement programme in Holstein‐Friesian cattle.     

Sperm penetration assay as an indicator of bull fertility

To predict the fertility of frozen-thawed bull spermatozoa, a sperm penetration assay (SPA) using zona-free hamster oocytes was optimized, and the assay results were compared with data from field fertility expressed as the non-return rate (NRR). To increase sperm penetration, the spermatozoa were pre-incubated and coincubated with oocytes in media containing various concentrations of heparin (0 to 50 μg/ml). Coincubation with 10 μg/ml heparin showed the highest sperm penetration (P<0.05); it is considered to be the optimized SPA method. Sperm fertility index values obtained from WSPA were significantly correlated with the historic average NRR of 46 bulls (P<0.01). To determine the normal range for SPA, we established the lower limits of the sperm fertility index and set the cut-off value at 2.55, at which point the NRR was more than 70%, using the receiver operating characteristic curve. The overall accuracy for the 46 bulls was 95.7% (44/46) for both the low and high NRR, with a sensitivity of 95.5% (21/22) and a specificity of 95.8%. This protocol would make it easier to discriminate bulls according to their sperm fertilizing ability.     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background: Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population. Results: We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM. Conclusions: These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Screening for bovine leukocyte adhesion deficiency,deficiency of uridine monophosphate synthase,complex vertebral malformation,bovine citrullinaemia,and factor XI deficiency in Holstein cows reared in Turkey

Background

Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins.

Methods

Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not.

Results

Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID).

Conclusion

This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.     

蒙贝利亚牛与荷斯坦牛在北方地区生长发育差异性比较

本研究对11头蒙贝利亚母牛和9头蒙贝利亚公牛的生长发育进行了跟踪测定,并与同期荷斯坦母牛的生长发育、产奶性能进行了对比分析。结果表明,蒙贝利亚母牛与荷斯坦母牛初生重无显著差异;4～18月龄,蒙贝利亚母牛体重均明显高于荷斯坦母牛同期体重,差异极显著（P〈0．01）。蒙贝利亚母牛与荷斯坦母牛日增重变化规律相似,lOB龄平均日增重最大,17月龄平均日增重最小。蒙贝利亚母牛各阶段体高、体长与荷斯坦母牛无显著差异,14月龄体高可达127cm,达到配种体高。蒙贝利亚母牛头胎305d产奶量为7241kg,对照组荷斯坦母牛头胎305d产奶量为9589kg,二者差异极显著（P〈0．01）。蒙贝利亚公牛初生重较大,至24月龄体重接近荷斯坦公牛同期体重。12~24月龄,荷斯坦公牛体高、体斜长、胸围均明显高于蒙贝利亚公牛,差异板显著（P〈0．01）;蒙贝利亚公牛初生重48kg,蒙贝利亚母牛初生重42kg,二者差异显著（P〈0．05）。蒙贝利亚公牛各阶段睾丸周径均显著高于荷斯坦公牛,差异极显著（P〈0．01）。     

A novel method for rapid and reliable detection of complex vertebral malformation and bovine leukocyte adhesion deficiency in Holstein cattle

Background

Complex vertebral malformation (CVM) and bovine leukocyte adhesion deficiency (BLAD) are two autosomal recessive lethal genetic defects frequently occurring in Holstein cattle, identifiable by single nucleotide polymorphisms. The objective of this study is to develop a rapid and reliable genotyping assay to screen the active Holstein sires and determine the carrier frequency of CVM and BLAD in Chinese dairy cattle population.

Results

We developed real-time PCR-based assays for discrimination of wild-type and defective alleles, so that carriers can be detected. Only one step was required after the DNA extraction from the sample and time consumption was about 2 hours. A total of 587 Chinese Holstein bulls were assayed, and fifty-six CVM-carriers and eight BLAD-carriers were identified, corresponding to heterozygote carrier frequencies of 9.54% and 1.36%, respectively. The pedigree analysis showed that most of the carriers could be traced back to the common ancestry, Osborndale Ivanhoe for BLAD and Pennstate Ivanhoe Star for CVM.

Conclusions

These results demonstrate that real-time PCR is a simple, rapid and reliable assay for BLAD and CVM defective allele detection. The high frequency of the CVM allele suggests that implementing a routine testing system is necessary to gradually eradicate the deleterious gene from the Chinese Holstein population.     

Genetic Description of Factor XI Deficiency in Holstein Semen in Western Japan

Factor XI deficiency was detected in Holstein cows and mummified foetuses in Japan; however, no report is available about the occurrence of Factor XI deficiency in Holstein semen in Japan. Five hundred cows in twelve dairy farms in Hiroshima Prefecture, Japan were under the study. Genomic DNA was extracted from the cows using a commercial DNA kits and screened to Factor XI mutation. Based on the information of the carrier cows found in the cattle population, four Holstein bulls were analysed for Factor XI mutation. DNA was extracted from bull's semen using phenol chloroform method. Extracted genomic DNA of the bull's semen was typed for Factor XI using specific polymerase chain reaction (PCR) primers. The resultant PCR was sequenced using big dye terminator sequencing method. The pedigree of the bulls was investigated. Furthermore, the inheritance of Factor XI mutation to next generation was estimated. Out of the 500 cows, five were heterozygous to Factor XI. Moreover, out of the four bulls, one was found to carry the mutation of Factor XI; it was also a complex vertebral malformation (CVM) carrier. In DNA sequencing, the insertion mutation of 76 bp of poly-adenine that characterizes the Factor XI deficiency was detected in the carrier bull as well as the carrier cows. Pedigree analysis of the carrier bull revealed that his father and mother ID were 2247419A and 14189172A, respectively, that originated from USA Holstein. Out of six daughter cows born to the carrier bull, one cow (16.6%) inherited Factor XI mutation, while three of them (50.0%) inherited CVM mutation. Autosomal recessive genes that affect cow's reproduction have a particular concern to dairy industry. To our knowledge this is the first report of Factor XI mutation in Holstein semen in Japan.     

荷斯坦牛脊椎畸形综合征分子诊断方法的建立与应用

脊椎畸形综合征(Complex Vertebral Malformation,CVM)是由常染色体上SLC35A3基因单碱基突变(G→T)引起的隐性遗传疾病,该基因隐性纯合(CV/CV)时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究自行设计检测CVM特异性PCR引物,扩增长度为173bp,然后利用PCR-SSCP方法对186个公牛样品和140个母牛样品进行了检测分析,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选。研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为11.3%和12.1%,并通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛"Penstate Ivanhoe Star"(USA.1441440,CV)。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以,有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

北京地区荷斯坦牛脊椎畸形综合征的调查研究

脊椎畸形综合征（Complex Vertebral Malformation,CVM）是由常染色体上SLC35A3基因单碱基突变（G→T）引起的隐性遗传疾病,该基因隐性纯合（CV/CV）时奶牛致死,但CVM携带者表现正常,所以CVM携带者公牛可以通过人工授精技术传播CVM缺陷基因。本研究利用PCR-SSCP方法对北京地区242头公牛样品和403头母牛样品进行了检测分析,研究结果表明,在所检测的样本中,荷斯坦种公牛和母牛CVM携带率分别为8.82%和5.71%,CVM基因频率分别为4.41%和2.85%。通过系谱追踪发现,CVM遗传缺陷的共同祖先是美国名牛＂Penstate Ivanhoe Star＂（USA.1441440,CV）。通过剔除CVM携带者公牛可以有效地控制CVM遗传缺陷的传播,但是,我国许多CVM携带者公牛冻精依然在商业化使用,所以有效地监控CVM携带者在奶牛群中的状况对CVM防控计划是有益的。     

我国荷斯坦青年公牛基因组选择效果分析

本研究基于我国荷斯坦奶牛基因组遗传评估和生产性能测定（DHI）结果,旨在分析我国荷斯坦公牛基因组选择的效果。选择1 686头既有基因组遗传评估成绩又有后裔测定成绩的荷斯坦公牛,利用2019年12月基因组遗传评估结果及其女儿的产奶和体型性状数据,通过R软件与Excel计算公牛基因组评估结果与公牛女儿表型数据间的相关性,对我国荷斯坦青年公牛基因组选择效果进行分析。相关性分析结果表明,荷斯坦公牛的基因组性能指数（GCPI）与后裔测定性能指数（CPI）呈正相关（r_s>0.3）,其中产奶量和体细胞评分的基因组育种值（GEBV）与估计育种值（EBV）呈较强的正相关（0.4 < r_s < 0.8）。对公牛女儿表型数据分析结果表明,女儿产奶量、乳蛋白率、乳脂率与肢蹄评分的表型值与公牛GEBV分组趋势一致,且公牛不同产奶性状GEBV组间的女儿性状表型值大部分达到极显著差异（P<0.01）;北京及上海地区公牛产奶性状、体细胞评分和肢蹄评分的GEBV分组与女儿表型值趋势较其他省市（地区）更一致,且GEBV高组与低组之间差值均高于其他省市（地区）。基于1 686头荷斯坦公牛基因组选择及其女儿表型数据的分析结果表明,我国荷斯坦公牛的基因组遗传评估准确性较好,其中产奶量、乳蛋白率、体细胞评分和肢蹄评分的表型数据更好地反映了基因组选择的效果;北京及上海地区较其他省市（地区）更能反映我国荷斯坦公牛基因组选择的效果。     

中国荷斯坦种公牛BLAD遗传缺陷的分子检测及系谱分析

本试验运用限制性片段长度多态性聚合酶链式反应（RFLP-PCR）方法,检测我国荷斯坦种公牛白细胞粘附缺陷（bovine leukocyte adhesion deficiency,BLAD）基因的携带频率。共检测了来自全国14个公牛站的587头种公牛,发现BLAD携带者8头,携带率为1.36%。对现有公牛系谱信息分析显示,携带者公牛来自美国、加拿大和中国,其中6头携带者公牛可以追溯到共同祖先Osborndale Ivanhoe。此外,本研究还对我国荷斯坦牛遗传缺陷的控制和携带者公牛的利用提出建议。     

部分中国荷斯坦种公牛脊柱畸形综合征携带状况的检测和分析

摘本研究旨在对部分中国荷斯坦种公牛脊柱畸形综合征(Complex vertebral malformation,CVM)致病基因的携带状况进行筛查.应用错配PCR突变分析技术(PCR mismatch amplification mutation assay,PCR-MAMA)建立了针对CVM致病基因的特异性检测方法.利用PCR-MAMA法检测了154头荷斯坦种公牛,发现了24头CVM阳性个体,阳性率为15.58％.结果显示,应对中国荷斯坦种公牛进行全面的针对CVM的检测.     

The Relationship Between Post‐Thaw Sperm DNA Integrity and Non‐Return Rate Among Norwegian Cross‐Bred Rams

With the aim of investigating the relationship between sperm DNA integrity and non‐return rate (NRR) among Norwegian cross‐bred rams, semen from 15 individuals was examined by flow cytometry. Sperm Chromatin Structure Assay (SCSA) quantifies the proportion of spermatozoa with denatured DNA after in situ acid treatment, and the four parameters % DFI, % HDS, MEAN DFI and SD DFI are all different measures of DNA denaturation and maturation. Field fertility, reported as NRR 25 days after insemination was based on all inseminations from a large‐scale breeding programme and supplied by the Norwegian Association of Sheep and Goat Farmers. From each ram, four straws from four different weeks of the breeding season were analysed, and the associations between 25‐day NRR and the mean of the four SCSA parameters were tested using a logistic regression model. The results revealed no association between fertility and % DFI or % HDS, while SD DFI and MEAN DFI showed a significant negative association with NRR. Further, the SCSA values varied significantly between ejaculates within ram among some of the rams in the study. However, no significant association was seen between these intra‐individual differences in sperm DNA integrity and NRR. In conclusion, this study suggests an association between sperm DNA integrity and NRR for rams. However, further research must be conducted to confirm these findings and determine whether sperm DNA assessments can be applied to predict ram fertility.     

Effect of acrosomal defects on fertility of bulls used in artificial insemination and natural breeding

Four bulls that produced spermatozoa with a high percentage of abnormal acrosomes were individually placed in pens with females for 21 days. Frozen semen from 2 of the bulls was used for artificial insemination. One of the bulls was placed in a competitive mating situation with normal bulls at pasture. First service pregnancy rates were determined by transrectal ultrasonography 28 days after bull removal from breeding pens, or after the last artificial insemination. The results of competitive mating at pasture were determined from breeding observations, the phenotypic characteristics of calves sired, and blood typing for parentage. The results of these studies suggest that bulls that produce a high percentage of spermatozoa with indented acrosomes may have normal fertility when used in artificial insemination or in single sire mating; however, their fertility may be low when breeding competitively with bulls with normal spermiograms.     

Fertility assessment through heterospermic insemination of flow-sorted sperm in cattle

The ability to assess fertility of bovine sperm accurately and rapidly would be very useful for research and applications to the cattle industry. Sperm motility and other in vitro tests of sperm normality are only partially correlated with fertility, and lengthy breeding trials are expensive and time consuming. Heterospermic insemination by mixing sperm from more than one male provides an in vivo method to assess relative fertility among bulls that can be economical and rapid. Sperm that had been flow-sorted and cryopreserved from four groups of four bulls were inseminated in all combinations of three bulls within groups into nonsuperovulated heifers or superovulated heifers. Embryos were collected nonsurgically between d 13.5 and 20 following estrus and evaluated for paternity by genotyping. Following determination of paternity, a heterospermic index was created for each bull using a maximum likelihood function. These indices ranged from 0.22 +/- 0.15 to 2.43 +/- 0.43 (mean = 1.00, with a higher value indicative of greater fertility). In all four groups, either the high- or low-fertility bull was identified (P < 0.05) using a total of 25 to 36 genotypable embryos from nonsuperovulated heifers. The heterospermic rankings of bulls were similar for single and superovulated heifers for one group of bulls, but dissimilar for a second group. Heterospermic insemination followed by genotyping of embryos proved to be efficacious for rapidly ranking fertility of flow-sorted sperm from bulls when females were not superovulated, but results were less clear when females were superovulated.     

Detection of quantitative trait loci affecting non-return rate in French dairy cattle

The purpose of this study was to map quantitative trait loci (QTL) influencing female fertility estimated by non-return rate (NRR) in the French dairy cattle breeds Prim'Holstein, Normande and Montbeliarde. The first step was a QTL detection study on NRR at 281 days after artificial insemination on 78 half-sib families including 4993 progeny tested bulls. In Prim'Holstein, three QTL were identified on Bos taurus chromosomes BTA01, BTA02 and BTA03 (p < 0.01), whereas one QTL was identified in Normande on BTA01 (p < 0.05). The second step aimed at confirming these three QTL and refining their location by selecting and genotyping additional microsatellite markers on a sub-sample of 41 families from the three breeds using NRR within 56, 90 and 281 days after AI. Only the three QTL initially detected in Prim'Holstein were confirmed. Moreover, the analysis of NRR within 56, 90 and 281 days after AI allowed us to distinguish two FF QTL on BTA02 in Prim'Holstein, one for NRR56 and one for NRR90. Estimated QTL variance was 18%, 14%, 11.5% and 14% of the total genetic variance, respectively, for QTL mapping to BTA01, BTA02 (NRR90 and NRR56) and BTA03.