Furosemide loading test in a case of homozygous solute carrier family 12, member 1 (SLC12A1) mutation (g.62382825G>A,p.Pro372Leu) in Japanese Black cattle

Hydrallantois is the excessive accumulation of fluid in the allantoic cavity in a pregnant animal and is associated with fetal death. We recently identified a recessive missense mutation in the solute carrier family 12, member 1 (SLC 12A1 ) gene (g.62382825G>A, p.Pro372Leu) that is associated with hydrallantois in Japanese Black cattle. Unexpectedly, we found a case of the homozygous risk‐allele for SLC 12A1 in a calf, using a PCR ‐based direct DNA sequencing test. The homozygote was outwardly healthy up to 3 months of age and the mother did not exhibit any clinical symptoms of hydrallantois. In order to validate these observations, we performed confirmation tests for the genotype and a diuretic loading test using furosemide, which inhibits the transporter activity of the SLC 12A1 protein. The results showed that the calf was really homozygous for the risk‐allele. In the homozygous calf, administration of furosemide did not alter urinary Na⁺ or Cl^? levels, in contrast to the heterozygote and wild‐type calves in which these were significantly increased. These results demonstrate that the SLC 12A1 (g.62382825G>A, p.Pro372Leu) is a hypomorphic or loss‐of‐function mutation and the hydrallantois with this mutation shows incomplete penetrance in Japanese Black cattle.     

Sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes

Bovine neonatal pancytopenia (BNP) is a bleeding and pancytopenic syndrome in neonatal calves, which recently emerged all over Europe. The present study tested whether antibodies directed against calf leukocytes are present in sera from known BNP dams. Sera from BNP dams (n=11) were combined with leukocytes from 11 calves (5 BNP survivors and 6 controls). After adding a fluorescein conjugated F(ab')(2) fragment of rabbit anti-bovine IgG (H&L) the level of antibody binding was measured by flow cytometry. As control groups both sera from dams from BNP affected (n=48) as from unaffected (n=54) herds were combined with leukocytes from the same calves. With sera from BNP dams, antibody binding could be visualised by immunofluoresence in both peripheral blood as in bone marrow smears. Mean fluoresence intensity values of all leukocyte subpopulations were significantly higher for the BNP dams compared to both control groups (P<0.01). BNP dams showed significantly more antibody binding on multiple leukocyte subpopulations of both BNP survivors and control calves and this from cut off values of MFI 100 onwards (P<0.01). The BNP survivor calves reacted significantly more often with sera from the BNP dams than the control calves (P<0.01). In conclusion the present study supports the hypothesis that BNP is an immune-mediated disease.     

Accounting for numbers of expressions of specific genotypes using a modified gene-flow method

The objective of this study was to develop a modified discounted gene-flow method and calculate numbers of cumulative discounted expressions (CDE) and value of using a Japanese Black cattle bull carrying specific alleles of interest. The discounted gene-flow method was modified to consider not only parent's genotypes but also the allele frequency in the herd. Input parameters representing a typical situation in a Japanese Black cattle herd were used to calculate the CDE and the value of using sires genotyped for recessive genes assuming biallelic loci (A and a) and a herd at Hardy–Weinberg equilibrium. The quantitative trait assumed was marbling and the effect of genotype aa was 1 Beef Marbling Standards (BMS) above that of an animal with either genotype Aa or AA. The effects of gene frequency and discount and survival rates on the CDE were determined for different genotypes of sires. Benefits from using either aa sires or Aa sires above that of using unknown sires at various gene frequencies were also determined. The CDE of aa sires were larger than those of Aa sires under all gene frequencies. The differences in the CDE between aa and Aa sires ranged from 34% at gene frequency of 0.7 to 71% at gene frequency of 0.01. An increase in the discount rates and a decrease in survival rates were associated with a decrease in expressions in cases using aa and Aa sires. The benefits of using aa sires were high when the gene frequency in the herd was 0.5. The benefit of aa sires were approximately 3023 yen more than that for unknown sires in the population when the gene frequency was 0.5. At gene frequencies higher than 0.5, use of Aa sires was not beneficial. This study has demonstrated how the gene frequencies in the herd and the genotypes of sires are critical in determining the benefits of using single recessive gene in the Japanese Black cattle.     

Genetic diagnosis of band 3 deficiency and sexing in bovine preimplantation embryos

Band 3 deficiency with hereditary spherocytosis and hemolytic anemia in Japanese black cattle, band 3(Bov.Yamagata), is caused by a total lack of band 3 protein with an autosomal dominant inheritance. Genotyping for band 3 deficiency and sexing were successfully achieved in biopsied embryo cells with efficiencies of 98.4% and 97.4%, respectively. Transfer of the embryo that was determined as homozygous for the mutant allele into a recipient cow resulted in the production of a fetus exhibiting the genotype and red cell phenotypes characteristic of band 3(Bov.Yamagata). These results demonstrate that our procedure is reliable and applicable to produce animals free from or homozygous for the mutant allele by breeding carrier animals.     

Genetic diagnosis of claudin-16 deficiency and sex determination in bovine preimplantation embryos

Renal tubular dysplasia is an autosomal recessively inherited disorder in Japanese black cattle that is due to deletion mutations in the claudin-16 gene and causes chronic renal failure and death of affected animals. Here, we report a multiplex-PCR procedure to determine the genotype for claudin-16 deficiency in preimplantation embryos. The presence or absence of the wild-type and mutant allele(s) was precisely detected with the multiplex-PCR using as little as 5 pg of genomic DNA from leukocytes. When biopsied embryo cells were examined for claudin-16 deficiency, 97.2% of genotypes were consistent with the PCR results obtained for the corresponding embryos. In addition, sexing of embryos by PCR was performed using an aliquot of DNA extracted from biopsied embryo cells, and determination of claudin-16 genotype and sex was successfully achieved with an efficiency of 91.7% for claudin-16 genotyping and 83.3% for sexing. The production of a 100-day fetus that was male and homozygous for claudin-16 deficiency, as expected from the analysis of biopsied embryo cells, gave evidence of the reliability and applicability of this procedure for preventing the transmission of this disease and for enabling advances in animal breeding.     

鲁西牛不同杂交组合生产性能比较分析

试验旨在探索鲁西黄牛与不同优良肉牛品种的杂交效果，提高鲁西黄牛的生产性能。选取111头健康公牛分为4组：A组为29头利木赞牛×鲁西牛杂交牛（利鲁牛）；B组为26头西门塔尔牛×鲁西牛杂交牛（西鲁牛）；C组为27头和牛×鲁西牛杂交牛（和鲁牛）；D组为29头纯种鲁西牛。在相同饲养管理条件下进行育肥及屠宰试验，并对育肥效果进行综合分析。结果显示，利木赞牛、西门塔尔牛、和牛对鲁西牛均有较好的杂交改良效果；平均日增重以西鲁牛最高，极显著高于其他3组牛（P<0.01）；宰前活重、胴体重、屠宰率均以西鲁牛最高，显著高于其他3组牛（P<0.05）；和鲁牛大理石花纹沉积能力最强；日平均采食量和日平均饲料成本在各组间差异均不显著（P>0.05）。综合分析，从产肉量来讲，西鲁牛最佳；从大理石花纹沉积能力来讲，和鲁牛最佳。     

Polymorphisms in growth hormone gene and their associations with calf weight in Japanese Black cattle

The objectives of this study were to detect effective genetic polymorphisms of bovine growth hormone (bGH) gene associated with calf weight in Japanese Black cattle. Fifty‐eight sires and 47 breeding cows were used to detect the polymorphisms in exons by single‐strand conformation polymorphism (SSCP). Four homozygous and six heterozygous SSCP genotypes were identified in exon 5. Although each single nucleotide polymorphism (SNP) had been reported, these genotypes were caused by three SNPs at the nucleotide positions 2141, 2277 and 2291. Four haplotypes C‐C‐A, G‐C‐A, C‐C‐C and G‐T‐A were newly identified. It was suggested that other haplotypes not detected in this study may not exist, considering the allele frequencies reported in Bos taurus and Bos indicus, and the migrating process of native Japanese cattle. Thereafter, we examined associations between the detected polymorphic sites in exon 5 by PCR – restriction fragment length polymorphism and calf weight using 53 breeding dams and 135 calves. The birth weights of calves with haplotype G‐C‐A are significantly lighter and calves' weights produced by cows with such haplotype are also lighter at 30 days old, using regression analysis. Although further research is necessary, these results may serve as a useful criterion to select breeding stocks, especially in maternal abilities.     

UTS2R gene polymorphisms are associated with fatty acid composition in Japanese beef cattle

Fatty acid composition of beef adipose tissue is one of its important traits because a high proportion of monounsaturated fatty acid is related to favorable beef flavor and tenderness. In this study, we searched polymorphisms in full length coding DNA sequence of urotensin 2 recepter and investigated the effects on fatty acid composition (C14:0, C14:1, C16:0, C16:1, C18:0, C18:1, C18:2, monounsaturated fatty acid, saturated fatty acid). Eight single nucleotide polymorphisms (SNP) were identified by sequence comparison among eight animals, including five Japanese Black and three Holstein cattle. One of these SNP (c.866C>T) was predicted to cause amino acid substitutions (P289L) and the other seven synonymous SNP, including c.267C>T, were presumed to be in linkage disequilibrium. Therefore we selected two SNP (c.267C>T and c.866C>T) for further analysis. We investigated associations between these genotypes and fatty acid composition in three Japanese Black populations (n = 560, 245 and 287) and a Holstein population (n = 202). Tukey‐Kramer's honestly significant difference test revealed that CC genotype in c.267C>T indicated lower C14:0 and higher C18:1 than the other genotypes in Japanese Black cattle and CC genotype in c.866C>T showed lower C16:1 than CT genotype in Holstein cattle (P < 0.05). These results suggested that these genotypes would contribute to production of high‐grade meat as selection markers in beef cattle.     

Plasma concentration of estrone sulfate during pregnancy in different breeds of Japanese beef cattle

Plasma concentrations of estrone sulfate in different breeds of Japanese beef cattle and the relationship between those concentrations and feto-placental growth were examined in order to assess the possibility of monitoring abnormal growth of the fetus. Blood samples were obtained from cows from day 90 of gestation to parturition. The plasma concentration of estrone sulfate was measured by direct enzyme immunoassay. From day 180 of gestation, the mean concentration of estrone sulfate increased gradually and it was drastically elevated after day 240 of gestation with the maximum at day 285. Plasma concentrations of estrone sulfate on day 240 of gestation was significantly increased in F(1) cows (Holstein Friesian and Japanese Black) compared with those in other breeds of cow. From day 270 to 278 of gestation, estrone sulfate concentrations of Holstein Friesian cows inseminated by Holstein Friesian differed from those inseminated by Japanese Black. In the cow with retained placenta, the plasma concentration of estrone sulfate reached plateau at day 240 of gestation and did not increase thereafter. There was no significant relationship between estrone sulfate concentration and duration of gestation, calf birth weight, weight of placenta or viability of newborn calves. These results indicate that changes of plasma estrone sulfate concentration in Japanese beef cattle are very similar to those in Holstein dairy cattle. They also suggest that the plasma concentration of estrone sulfate is associated with the breed of pregnant cow and that its concentration is also affected by calf birth weight depending on the breed of bull. It seems possible to predict the incidence of retained placenta but not the calf birth weight and viability of newborn calves in Japanese beef cattle.     

Identification of leptin gene polymorphisms associated with carcass traits and fatty acid composition in Japanese Black cattle

Previous studies have indicated that some leptin gene polymorphisms were associated with economically important traits in cattle breeds. However, polymorphisms in the leptin gene have not been reported thus far in Japanese Black cattle. Here, we aimed to identify the leptin gene polymorphisms which are associated with carcass traits and fatty acid composition in Japanese Black cattle. We sequenced the full‐length coding sequence of leptin gene for eight Japanese Black cattle. Sequence comparison revealed eight single nucleotide polymorphisms (SNPs). Three of these were predicted to cause amino acid substitutions: Y7F, R25C and A80V. Then, we genotyped these SNPs in two populations (JB1 with 560 animals and JB2 with 450 animals) and investigated the effects on the traits. Y7F in JB1 and A80V in JB2 were excluded from statistical analysis because the minor allele frequencies were low (< 0.1). Association analysis revealed that Y7F had a significant effect on the dressed carcass weight in JB2; R25C had a significant effect on C18:0 and C14:1 in JB1 and JB2, respectively; and A80V had a significant effect on C16:0, C16:1, C18:1, monounsaturated fatty acid and saturated fatty acid in JB1. The results suggested that these SNPs could be used as an effective marker for the improvement of Japanese Black cattle.     

Performance of using opposing homozygotes for paternity testing in Japanese Black cattle

Genome-wide single nucleotide polymorphism (SNP) markers in Japanese Black cattle enable genomic prediction and verifying parent–offspring relationships. We assessed the performance of opposing homozygotes (OH) for paternity testing in Japanese Black cattle, using SNP genotype information of 50 sires and 3,420 fattened animals, 1,945 of which were fathered by the 50 genotyped sires. The number of OH was counted for each sire–progeny pair in 28,764 SNPs with minor allele frequencies of ≥0.05 in this population. Across all pairs of animals, the number of OH tended to increase as the pedigree-based coefficient of relationship decreased. With a threshold of 288 (1% of SNPs) for paternity testing, most sire–progeny pairs were detected as true relationships. The frequency of Mendelian inconsistencies was 2.4%, reflecting the high accuracy of pedigree information in Japanese Black cattle population. The results indicate the utility of OH for paternity testing in Japanese Black cattle.     

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.     

Pedigree analysis of factor XI deficiency in Japanese black cattle

Using a DNA-based diagnostic test for factor XI deficiency in Japanese black cattle, we surveyed 123 cattle (42 sires and 81 dams) in Gifu and Hyogo prefectures, and calculated gene frequencies. In sires, we drew up the pedigree network of the cattle with the factor XI deficiency. Results showed that the mutated allele of factor XI deficiency was retroactive in at least 6 or more generations of sires. Frequencies of the mutant gene were higher at 26.4% in total, and at 33.3% in sires. All 7 cattle with the homozygote of mutated allele were clinically normal, and showed no bleeding episodes. The mutated allele of factor XI deficiency might be widespread among Japanese black cattle.     

Growth of Holstein calves from birth to 90 days: the influence of dietary zinc and BLAD status

The main objective of this study was to describe Holstein neonatal growth and development as influenced by dietary zinc supplementation and the CD18 genotype, both of which may affect immune competence. Holstein calves (n = 421), after being fed colostrum, were brought to a calf facility, randomly assigned to one of four zinc supplementation groups (control at 40 mg Zn/kg DM or the control diet supplemented with an additional 60 mg Zn/kg DM provided as either zinc sulfate, zinc lysine, or zinc methionine), weighed, and measured for morphometric growth parameters. Measurements were repeated at 30, 60, and 90 d. Calves were also genotyped for the presence of the mutant D128G CD18 allele, which, if present in two copies, causes bovine leukocyte adhesion deficiency. Zinc supplementation above 40 mg Zn/kg DM, regardless of the chemical form, did not accelerate growth (P > 0.25). Further, overall calf growth performance was not suppressed or improved (P > 0.4) in calves heterozygous at the CD18 locus relative to calves homozygous for the normal CD18 allele, although genotype negatively affected some morphometric measurements (P < 0.05). Using these data, quadratic models of early growth were generated as a preliminary step to develop growth criteria that will allow producers, veterinarians, and animal scientists to identify poor growth performance early in neonatal life. Such criteria provide the basis for tools to improve economic performance.     

Renal reabsorption of magnesium and calcium by cattle with renal tubular dysplasia

The concentrations of magnesium and calcium in the serum and urine and their rates of clearance were determined in cattle with renal tubular dysplasia, an autosomal recessive hereditary disease associated with a deletion of the paracellin-1 gene in Japanese Black cattle. There were no significant differences in the serum or urine magnesium concentrations between normal cattle and cattle which were heterozygous or homozygous for the condition. Serum calcium concentrations tended to be lower in the homozygous cattle, and the serum creatinine and urea nitrogen concentrations were significantly higher in the homozygous cattle. The ratio of magnesium:creatinine and the fractional excretion of magnesium were higher in cattle with the disease than in normal cattle. There were no significant differences in urine calcium concentration, the calcium:creatinine ratio, and fractional excretion of calcium between normal cattle and cattle which were homozygous or heterozygous for the condition. The creatinine clearance was significantly lower in the homozygous cattle than in normal cattle. The clearance, excretion rate, reabsorption rate and reabsorption rate:clearance ratio of magnesium in cattle with renal tubular dysplasia were significantly lower than in normal cattle. The clearance rate and reabsorption rate of calcium were also significantly lower in the affected cattle, but the excretion rate and reabsorption rate:clearance of calcium were not different between the normal cattle and the cattle homozygous for the condition. In cattle with the condition the rate of reabsorption of magnesium by the kidneys was low, but the rate of reabsorption of calcium was normal.     

Frequency of bovine Nramp1 (Slc11a1) alleles in Holstein and Zebu breeds

Natural resistance against brucellosis in cattle is linked to the Nramp1 gene, which encodes a divalent cation transporter that localizes in the phagolysosome membrane in macrophages. Nramp1 gene in mouse plays a critical role in innate immunity favoring bacterial killing by macrophages in addition to its influence on adaptative immunity. Polymorphisms at the bovine Nramp1 3' untranslated region (3'UTR), detectable by Single Strand Conformational Analysis (SSCA), are associated with natural resistance against brucellosis. Such polymorphisms are associated with variation in the number of GT repeats. This study compared the frequency of Nramp1 3'UTR polymorphisms between Zebu and European bovine breeds. Eighty-one Holsteins (Bos taurus taurus) and 167 Zebu (Bos taurus indicus), including the following breeds: Nelore (n=95), Guzerá (n=37), and Gir (n=35), totaling 248 pure breed cattle studied. DNA extraction was performed using the guanidium protocol and genotyping was performed by SSCA. DNA from cattle considered genotypically resistant to brucellosis resulted in a single band (homozygous) with 175bp, corresponding to the 3'UTR with 13 GT pairs (GT13), whereas DNA from genotypically susceptible cattle generated one single band with 177bp (homozygous GT14) or double bands with both 175 and 177bp, or 175 and 179bp (heterozygous GT13/GT14 or GT13/GT15, respectively). A marked difference in the frequency of alleles was detected between the Zebu and Holstein cattle. Holsteins had an extremely homogeneous genotype, with 100% of the individuals with a GT13 genotype. In sharp contrast the Nelore breed had the most heterogeneous genotype with four allelic combinations, namely, homozygous GT13, homozygous GT14, heterozygous GT13/GT14, and heterozygous GT13/GT15. When the Zebu breeds were compared to each other, the only significant difference observed was the frequencies of the genotypes GT13 and GT14 between the Nelore and Guzerá breeds. The knowledge of allelic frequencies in different breeds of cattle may prove to be very useful in the future for planning breeding strategies for selection of resistant cattle.     

Genetic variability of Wagyu cattle estimated by statistical approaches

The genetic evaluation of economically important traits utilizes estimates of genetic variability, which are represented by heritability. This review summarizes the published heritabilities of traits estimated in Wagyu cattle. Two different mean heritabilities, unweighted and weighted by standard errors, were calculated. In Japanese Black cattle, the average unweighted and weighted direct heritabilities of birth weight were 0.35 and 0.28, respectively, whereas the respective maternal heritabilities were 0.17 and 0.07. The mean unweighted heritability of calf market weight was estimated to be 0.30 in Japanese Black cattle. The mean unweighted heritability of daily gain during performance testing was 0.29 in Japanese Black and 0.40 in Japanese Shorthorn cattle. In Japanese Black cattle, the unweighted mean heritability was 0.48 for carcass weight, 0.46 for rib‐eye area, 0.38 for rib thickness, 0.39 for subcutaneous fat thickness, and 0.55 for marbling. The mean weighted heritability of the calving interval was low, and estimated to be 0.05. In general, the heritabilities estimated in Wagyu cattle were similar to those estimated in other beef breeds.     

Allele frequencies of the extension locus encoding the melanocortin-1 receptor in Japanese and Korean cattle

In order to estimate the influence of the Extension (E) locus in cattle coat color, the melanocortin‐1 receptor (MC1R) gene in Japanese Black, Japanese Brown and Korean (Hanwoo) cattle were sequenced. The sequences of the coding region revealed three alleles (E^D, E⁺ and e), which were previously reported. Polymerase chain reaction‐restriction fragment length polymorphism was performed to investigate the gene frequencies of the three breeds. Japanese Black was almost composed of E^D and E⁺ individuals, E^D = 0.481 and E⁺ = 0.514, and no homozygous e/e, therefore that is consistent with the hypothesis that E^D and E⁺ induce black pigment synthesis. Allele frequencies between Japanese Brown and Hanwoo were obviously different; however, recessive red e allele frequency was 0.038 for Japanese Brown and 0.948 for Hanwoo, even though both breeds have quite similar coat colors (ranging from yellowish brown to dark brown including a red coat color). This result suggested that other genes are also associated with a coat color of red and brown in cattle.     

Genetic structure and relationships of 16 Asian and European cattle populations using DigiTag2 assay

In this study, we genotyped 117 autosomal single nucleotide polymorphisms using a DigiTag2 assay to assess the genetic diversity, structure and relationships of 16 Eurasian cattle populations, including nine cattle breeds and seven native cattle. Phylogenetic and principal component analyses showed that Bos taurus and Bos indicus populations were clearly distinguished, whereas Japanese Shorthorn and Japanese Polled clustered with European populations. Furthermore, STRUCTURE analysis demonstrated the distinct separation between Bos taurus and Bos indicus (K=2), and between European and Asian populations (K=3). In addition, Japanese Holstein exhibited an admixture pattern with Asian and European cattle (K=3‐5). Mongolian (K=13‐16) and Japanese Black (K=14‐16) populations exhibited admixture patterns with different ancestries. Bos indicus populations exhibited a uniform genetic structure at K=2‐11, thereby suggesting that there are close genetic relationships among Bos indicus populations. However, the Bhutan and Bangladesh populations formed a cluster distinct from the other Bos indicus populations at K=12‐16. In conclusion, our study could sufficiently explain the genetic construction of Asian cattle populations, including: (i) the close genetic relationships among Bos indicus populations; (ii) the genetic influences of European breeds on Japanese breeds; (iii) the genetic admixture in Japanese Holstein, Mongolian and Japanese Black cattle; and (iv) the genetic subpopulations in Southeast Asia.     

Bovine leukemia virus genotype surveillance in cattle at a slaughterhouse in Aichi Prefecture,Japan, in 2019 using polymerase chain reaction combined with restriction fragment length polymorphism

Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) is commonly used for genotyping bovine leukemia virus (BLV) in slaughterhouses. However, unclassified BLV genotypes have been sporadically reported. To assess the current status of BLV genetic characterization in cattle, PCR-RFLP was performed on blood samples of 170 cattle (84 Japanese Black, 60 Japanese Black x Holstein, and 26 Holstein) from 17 farms (5 prefectures) at a slaughterhouse in Aichi Prefecture in 2019. A total of 65 samples (38.2%) were BLV positive, and genotype 1 was the most predominant (56/65 samples), followed by genotypes 3 (6 samples) and 5 (1 sample), and two unclassified samples. No relationship between the genotypes and breeds was observed. Sequence and phylogenetic analyses demonstrated that unclassified BLV genotypes clustered with genotype 1 sequences were, therefore, not new genotypes.