牛脊柱畸形综合征检测方法的建立与应用

牛脊柱畸形综合征(Complex vertebral malformation, CVM)是近年来新发现的致死性牛常染色体隐性遗传缺陷病。由于编码UDP-N-乙酰葡糖胺载体的SLC35A3基因发生G→T的突变而引起本病的发生, 可引起胎牛死胎、流产、早产。为了解我国正常的荷斯坦牛(黑白花奶牛)的CVM携带和发生情况, 建立、应用创造酶切位点PCR(Created restriction site PCR, CRS-PCR)、等位基因特异性PCR(Allele-specific polymerase chain reaction, AS-PCR)检测方法检测了表型正常的436头荷斯坦母牛和93头荷斯坦公牛, 检测到3头CVM携带者, 其中杂合母牛1头, 杂合公牛2头, 携带率分别为0.60%、2.20%。此方法简便、可靠, 为奶牛CVM有害基因的分型和筛选提供了新的方法和思路, 为我国奶牛的分子选育提供了可靠的理论依据。     

Complex vertebral malformation in Holstein cows in Japan and its inheritance to crossbred F1 generation

Complex vertebral malformation (CVM) is an autosomal recessive inherited defect in the Holstein breed. It causes intra-uterine mortality leading to repeat breeding and involuntary culling of cows. This study was carried out to show the prevalence of CVM carriers among Holstein cows in five dairy farms in Hiroshima Prefecture, South Western Japan and whether the defect could be inherited to crossbred F1 generation, when Japanese Black semen was used to inseminate a Holstein cow. Two hundreds Holstein cows were used in the study. Blood samples were collected from all cows in a heparinized tubes and genomic DNA was extracted with a commercial kit. Apart of the bovine solute carrier family 35 member 3 (SLC35A3) gene around the suspected mutation (G to T) was amplified with an allele specific polymerase chain reaction (AS-PCR). For DNA sequencing, PCR products of 522 bp were obtained from the genomic DNA of the cows. Out of 200 examined cows, 26 cows (13.0%) were found to be CVM carriers. Wild type cows showed amplification with the G-specific primer pair but not with the T-specific primer pair, while carrier ones showed amplification of both the G- and T-specific primers, exhibited two DNA bands of 395 bp. Based on the clinical history of the cows, lowered reproductive performance was noticed in carrier ones. Out of four crossbred F1 calves (Holstein x Japanese Black), two were CVM carrier. Crossbred F1 could inherit the CVM defect when Japanese Black semen used to inseminate a carrier Holstein cow. In conclusion, the study reports the occurrence of CVM among Holstein cows in Hiroshima Prefecture, Japan; moreover, it describes the first cases of CVM in crossbred F1 generation.     

中国荷斯坦牛CVM的基因检测及其与产奶性状的关联分析

脊椎畸形综合征(Complex vertebral malformation, CVM)是由位于牛第3号染色体(BTA3)的SLC35A3基因外显子4的一个单碱基突变(G559T)所致。该致病基因在世界许多国家的荷斯坦牛群中都有一定的比例。文章对北京地区38头优秀种公牛进行分析, 发现了4头携带者, 进而检测了这些携带者公牛的555头女儿的基因型, 其中携带者占检测母牛数的44.0%。此外, 关联分析结果表明, 携带者母牛与非携带者母牛的生产性能之间存在显著差异(P<0.01)。携带者母牛的5个产奶性状育种值均显著高于非携带者, 泌乳持续力和体细胞评分SCS的育种值也比非携带者略高。CVM致病基因可能与BTA3上影响产奶性状的QTL或基因连锁。因此, 建议生产中对CVM携带者进行逐步淘汰     

Detection of Haplotypes Associated with Prenatal Death in Dairy Cattle and Identification of Deleterious Mutations in GART,SHBG and SLC37A2

The regular decrease of female fertility over time is a major concern in modern dairy cattle industry. Only half of this decrease is explained by indirect response to selection on milk production, suggesting the existence of other factors such as embryonic lethal genetic defects. Genomic regions harboring recessive deleterious mutations were detected in three dairy cattle breeds by identifying frequent haplotypes (>1%) showing a deficit in homozygotes among Illumina Bovine 50k Beadchip haplotyping data from the French genomic selection database (47,878 Holstein, 16,833 Montbéliarde, and 11,466 Normande animals). Thirty-four candidate haplotypes (p<10⁻⁴) including previously reported regions associated with Brachyspina, CVM, HH1, and HH3 in Holstein breed were identified. Haplotype length varied from 1 to 4.8 Mb and frequencies from 1.7 up to 9%. A significant negative effect on calving rate, consistent in heifers and in lactating cows, was observed for 9 of these haplotypes in matings between carrier bulls and daughters of carrier sires, confirming their association with embryonic lethal mutations. Eight regions were further investigated using whole genome sequencing data from heterozygous bull carriers and control animals (45 animals in total). Six strong candidate causative mutations including polymorphisms previously reported in FANCI (Brachyspina), SLC35A3 (CVM), APAF1 (HH1) and three novel mutations with very damaging effect on the protein structure, according to SIFT and Polyphen-2, were detected in GART, SHBG and SLC37A2 genes. In conclusion, this study reveals a yet hidden consequence of the important inbreeding rate observed in intensively selected and specialized cattle breeds. Counter-selection of these mutations and management of matings will have positive consequences on female fertility in dairy cattle.     

Effects of Complex Vertebral Malformation on Fertility in Swedish Holstein Cattle

Complex vertebral malformation (CVM) is an autosomal recessive inherited defect in the Holstein breed. It causes intra-uterine mortality through the entire gestation period leading to repeat breeding and involuntary culling of cows and thereby economic losses. The defect was first reported in Denmark in 1999 and a direct DNA test for the defect has been available since February 2001. The aim of this study was to investigate if Holstein bulls heterozygous for the CVM gene had reduced reproductive performance, measured as non-return rate (NRR) and in a daughter fertility index. All genotyped Swedish Holstein bulls born between 1995 and 1999 were included. Altogether 228 bulls were analysed, of which 53 bulls, i.e. 23%, were confirmed CVM carriers. A statistically significant difference between carriers and non-carriers in the relative breeding value for NRR was observed for 168 days NRR (101.1 ± 0.9 vs. 103.1 ± 0.6, p < 0.05). There was no difference for 28 days NRR whereas the difference approached significance for 56 days NRR. No significant effect of the paternal CVM genotype on the daughter fertility index was shown probably due to the complexity of traits this index is composed of. In conclusion, the study showed that carriers of the CVM defect have an inferior NRR compared with non-carriers.     

荷斯坦奶牛脊柱畸形综合征的研究进展

荷斯坦奶牛脊柱畸形综合征是近年新发现的一种常染色体隐性遗传病,该病对纯合子胎儿是致死性的。当荷斯坦牛的育种核心群中携带者达到一定比例,脊柱畸形综合征就会对奶业造成巨大损失。文章综述了脊柱畸形综合征的主要症状和影响、致病基因及检测方法的研究进展以及我国潜在的荷斯坦奶牛脊柱畸形综合征携带者的状况。     

Prevalence of complex vertebral malformation carriers among Polish Holstein-Friesian bulls

An increasing number of Holstein calf births exhibiting vertebral deformations has been detected in Denmark since 1999 by a program monitoring the incidence of genetic diseases. Pedigree analysis demonstrated that the affected calves originated from a family afflicted by an autosomally recessively inherited complex vertebral malformation (CVM) syndrome. To determine the actual carrier frequency of the CVM-determining mutation in a population of Polish Holstein-Friesian (=Polish Black-and-White) cattle, we examined 202 proven bulls (active in 2001–2005) used by 4 domestic artificial insemination companies and 403 unproven bulls (under evaluation for breeding value). Out of the 605 bulls examined, 150 T/G heterozygotes were diagnosed, including 118 that were sons of known CVM carriers. Identification of a gene polymorphism in a bovine solute carrier family 35 member 3, termedSLC35A3, was conducted with the use of a new PCR-SSCP method (polymerase chain reaction - single stranded conformation polymorphism), which - due to its ease of use and high reliability - can be applied in widespread screening programs aimed at reducing the incidence of the CVM defect.     

Effects of sire and dam genotype for complex vertebral malformation (CVM) on risk of return-to-service in Holstein dairy cows and heifers

The emergence of a new hereditary disease, called "Complex vertebral malformation" (CVM), has been described in Holstein cattle population. This paper studied the incidence of mating concerning Holstein dairy cattle with CVM in Brittany and the possible influences of CVM status for sires and dams on return-to-service at different intervals post-service in cows and heifers. It was carried out based on a set of data for first and second inseminations between 1998 and 2001 in cows (n=530,538) and heifers (n=248,140). Incidence of matings between CVM gene carriers, between a carrier bull and a non-carrier cow and between a non-carrier bull and a carrier cow were estimated to be 1.4, 10.6 and 9%, respectively (1.1, 9.3 and 10.9%, respectively in heifers). Compared to CVM-free mating, the relative risk of return-to-service was increased when the sire was a CVM carrier and the dam was at risk of being a carrier, especially for late return (>25 days post-service). When the sire alone was a carrier, the relative risk was increased whenever the return occurred at a low but significant level. Following mating between a non-carrier bull and a dam at risk of being a carrier, the risk of return-to-service decreased at a low but significant level, whatever the interval in the heifers but only for 19-25 days intervals in the cows.     

No incidence of DUMPS carriers in Polish dairy cattle

DUMPS (Deficiency of Uridine Monophosphate Synthase) is a hereditary recessive disorder in Holstein cattle causing early embryo mortality during its implantation in the uterus. The only way to avoid the economic losses is early detection of DUMPS carriers. Because American Holstein semen has been intensively imported to Poland since 1970, there was a risk that DUMPS could have spread in Polish dairy cattle. In our study, 2209 dairy cattle of the Polish Holstein breed have been screened by the DNA test. The dominant group was young bulls entering the testing program (1171) and proven bulls (781). They represented all sires entering Polish breeding programs between 1999 and 2003. Also, 257 sire dams were included in the screening program. No DUMPS carrier has been found. Our results then indicate that the population of dairy cattle reared in Poland is free from DUMPS. Because of the economical significance of the DUMPS mutation and its recessive mode of inheritance, attention has to be paid to any case of a bull having in his origin any known DUMPS carrier. Such a bull should be tested and if positive eliminated from the active population. Also, young bulls (testing bulls) should be screened for DUMPS if in their progeny a high incidence of embryo mortality is observed and their genealogy cannot exclude their relatedness to any DUMPS carriers.     

Prevalence of Carriage of Shiga Toxin-Producing Escherichia coli Serotypes O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28 among Slaughtered Adult Cattle in France

The main pathogenic enterohemorrhagic Escherichia coli (EHEC) strains are defined as Shiga toxin (Stx)-producing E. coli (STEC) belonging to one of the following serotypes: O157:H7, O26:H11, O103:H2, O111:H8, and O145:H28. Each of these five serotypes is known to be associated with a specific subtype of the intimin-encoding gene (eae). The objective of this study was to evaluate the prevalence of bovine carriers of these “top five” STEC in the four adult cattle categories slaughtered in France. Fecal samples were collected from 1,318 cattle, including 291 young dairy bulls, 296 young beef bulls, 337 dairy cows, and 394 beef cows. A total of 96 E. coli isolates, including 33 top five STEC and 63 atypical enteropathogenic E. coli (aEPEC) isolates, with the same genetic characteristics as the top five STEC strains except that they lacked an stx gene, were recovered from these samples. O157:H7 was the most frequently isolated STEC serotype. The prevalence of top five STEC (all serotypes included) was 4.5% in young dairy bulls, 2.4% in young beef bulls, 1.8% in dairy cows, and 1.0% in beef cows. It was significantly higher in young dairy bulls (P < 0.05) than in the other 3 categories. The basis for these differences between categories remains to be elucidated. Moreover, simultaneous carriage of STEC O26:H11 and STEC O103:H2 was detected in one young dairy bull. Lastly, the prevalence of bovine carriers of the top five STEC, evaluated through a weighted arithmetic mean of the prevalence by categories, was estimated to 1.8% in slaughtered adult cattle in France.     

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.     

利用PCR-RFLP检测中国荷斯坦牛遗传缺陷——瓜氨酸血症

瓜氨酸血症(Citrullinemia)是荷斯坦牛尿素循环发生代谢紊乱的一种常染色体隐性遗传缺陷。精氨酸琥珀酸合成酶基因外显子5发生突变(C-T)对这一紊乱负责。本研究应用PCR-RFLP方法和DNA测序技术检测济南市周边120头荷斯坦母牛和山东奥克斯生物技术有限公司种公牛站50头荷斯坦公牛的精氨酸琥珀酸合成酶基因外显子5。结果发现,所检测的公牛未发现瓜氨酸血症突变基因携带者,母牛中有2头为携带者,携带频率为1.18%。     

Identification of Bovine Leucocyte Adhesion Deficiency (BLAD) Carriers in Holstein and Brown Swiss AI Bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene, which is responsible for this disease, has recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101-bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation was inherited by him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI, and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

中国荷斯坦牛白细胞黏附缺陷症PCR-RFLP检测方法的研究

本试验根据已知牛染色体上CD18编码基因序列设计引物,提取牛血液和精液DNA,可扩增出338bp的DNA片段,将PCR产物克隆到pMD18-T载体中,对阳性重组质粒进行测序,确定为牛的CD18基因。由于CD18基因的383位碱基由A变为G,而引起牛白细胞黏附缺陷症(BLAD),通过对济南市11个奶牛场356头奶牛及53头荷斯坦种公牛进行了BLAD的PCR-RFLP检测,共检出3头杂合母牛(携带者),占检测母牛群的0.84%,在荷斯坦公牛中只检测到一种基因型,没有发现隐性突变基因的携带者。     

Identification of bovine leucocyte adhesion deficiency (BLAD) carriers in Holstein and Brown Swiss AI bulls in Iran

BLAD is a hereditary disease in Holstein dairy cattle. The defective allele of CD18 gene which is responsible for this disease has a recessive inheritance. The recessive homozygous form (BL/BL) is lethal and since carrier animals have viability, BLAD frequency increases by use of carrier bulls in Artificial Insemination (AI). BLAD carriers can be detected easily by means of polymerase chain reaction followed by restriction analysis of the amplicons. In this study DNA samples from Holstein (n = 30) and Brown Swiss (n = 10) bulls from Abbas Abad AI center (Khorasan state of Iran) were analysed. A 101 bp fragment from the polymorphic region of CD18 gene located on chromosome 1 was amplified by PCR. Restriction enzymes TaqI and HaeIII were used to identify genotypes. Digestion products were screened by electrophoresis on 8% non-denaturing polyacrylamide gel and visualized by ethidium bromide staining. Frequencies of BL/TL (carrier) genotypes in Holstein and Brown Swiss bulls were 3.33% and 0%, respectively. Our pedigree studies of the carrier bull in this experiment revealed that the mutation inherited to him from Hawkeye bull (CANM 369995, BL). Although the elimination of BLAD-carrier bulls from the Holstein world would be the most efficient method to control this genetic disorder, many BLAD-carrier bulls are still listed commercially for AI and BLAD is still occurring in Iran. Monitoring the prevalence of BLAD-carriers in random selected herds may be helpful in judging the effectiveness of the BLAD-control program.     

Influence of sire and sire breed (Gyr versus Holstein) on establishment of pregnancy and embryonic loss in lactating Holstein cows during summer heat stress

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress; Bos taurus indicus (B. t. indicus) cattle and embryos are more thermotolerant than Bos taurus taurus (B. t. taurus). In the present study, the effects of sire and sire breed on conception and embryonic/fetal loss rates of lactating Holstein cows during the Brazilian summer were determined. In Experiment 1, cows (n=302) were AI after estrus detection or at a fixed-time with semen from one Gyr (B. t. indicus) or one Holstein sire (B. t. taurus). Pregnancy was diagnosed 80 days after AI. In Experiment 2, cows (n=811) were AI with semen from three Gyr and two Holstein sires. Pregnancy was diagnosed at 30-40 and at 60-80 days after AI. Cows diagnosed pregnant at the first examination but non-pregnant at the second were considered as having lost their embryo or fetus. Data were analyzed by logistic regression. The model considered the effect of sire within breed, sire breed, days postpartum, period of lactation, and AI type (AI after estrus versus fixed-time). There was no effect of the AI type, days postpartum or milk production on conception or embryonic loss rates. The use of Gyr bulls increased pregnancy rate when compared to Holstein bulls [9.1% (60/657) versus 5.0% (23/456), respectively, P=0.008; data from Experiments 1 and 2 combined]. Additionally, in Experiment 2, cows inseminated using semen from sire #4 (Gyr) had lower embryonic loss (10%) when compared with other B. t. indicus (35.3% and 40%) or B. t. taurus sires (18.2% and 38.5%, P=0.03). In conclusion, the use of B. t. indicus sires may result in higher conception rates in lactating Holstein cows during summer heat stress. Moreover, sire can affect embryonic loss and selection of bulls according to this criterion may result in higher parturition rates in lactating Holstein cows.     

Whole‐Genome association analysis of susceptibility to paratuberculosis in Holstein cattle

The objective of this study was to identify genetic markers and genomic regions associated with susceptibility to Mycobacterium avium ssp. paratuberculosis (MAP) infection in Holstein cattle. Associated single nucleotide polymorphisms (SNPs) were identified by genotyping 521 MAP‐infected Holstein cows and comparing SNP allele frequencies of these infected cows with allele frequencies estimated from specific reference populations. Reference population allele frequency estimates used Holstein sire genotype data and were weighted estimates based on sire usage within the population in question. The 521 infected cows were 233 and 288 cows from two resource populations of approximately 5000 cows each, collected independently. Population 1 was comprised primarily of daughters of twelve Holstein artificial insemination sires used heavily within the US dairy cattle population. Samples were obtained from 300 co‐operating commercial dairy herds throughout the US and were tested by both MAP faecal culture and blood‐enzyme‐linked immunosorbent assay (ELISA). Population 2 consisted of dairy cattle from six co‐operating dairy herds in Wisconsin, with all animals in the herds tested by blood enzyme‐linked immunosorbent assay (ELISA) for MAP infection. Genotyping was performed with the Illumina Bovine SNP50 Bead Chip, providing genotypes for 35 772 informative SNPs. Data from the two resource populations were analysed both in separate and combined analyses. The most significant autosomal markers from the individual and combined analyses (n = 197, nominal P < 0.001) were used in a stepwise logistic regression analysis to identify a set of 51 SNPs that could be used as a predictor of genetics for Holstein cattle susceptibility to MAP infection.     

Variation of the BoLA-DRB3 gene in dairy cattle and its effect on the viability parameters

Genotyping of the BoLA-DRB3 alleles was performed in dairy cattle of Krasnodar krai and Holstein stud bulls. Loss of heterozygosity, which decreased the reproductive parameters, was observed. It was proposed that stud bulls be selected on the basis of their genotyping at the BoLA-DRB3 gene to prevent further decay of cattle viability.     

Diagnosis of trichomoniasis in pregnant cows by culture of cervical-vaginal mucus

The objective of this study was to evaluate the effectiveness of Cervical-Vaginal Mucus (CVM) cultures for isolating $\text{Trichomonas fetus}$ from pregnant cows. Cervical-vaginal mucus was cultured from sixty-eight cows averaging 98 days of gestation. $\text{Trichomonas fetus}$ was isolated from 7 of 36 cows in groups exposed to bulls found to be infected at the end of the breeding season. The remaining 32 cows which were bred to bulls found not to be infected at the end of the breeding season were negative. Seven of the cows serviced by infected bulls aborted. Three of these had been positive and 4 negative on culture. The incidence of abortion was determined by examination 81 days after the previous examination and CVM culture. During the same interval there were no abortions in the 32 cows served by non-infected bulls. Culture of CVM identified infected groups of cows which had a high risk for abortion.     

Association of IL8 -105G/A with Mastitis Somatic Cell Score in Chinese Holstein Dairy Cows

The single nucleotide polymorphisms (SNPs) in the 5′ upstream of bovine IL8 gene were investigated in 810 Chinese Holstein cows from 35 bull families in a dairy farm in Shanghai using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The Real-time PCR and Western blot were used to detect the mRNA and protein levels of genotype Chinese Holstein dairy cows. The results showed that one SNP -105G>A was detected, designating three genotypes (GG, GA and AA) with respective frequencies of 0.38, 0.46, and 0.16. The significant association of the SNP -105G>A with somatic cell score (SCS) was identified. Genotype GG had a significantly lower SCS than genotype GA or AA (P < 0.01), and the relative mRNA expression and protein level of GG was found to be the highest. These results suggest that the genotype GG may be a useful genetic marker for mastitis resistance selection and breeding in Chinese Holstein dairy cows.