Polymorphism in promoter region of growth hormone receptor is associated with potential production capacity of insulin‐like growth factor‐1 in pre‐pubertal Holstein heifers

Insulin‐like growth factor‐1 (IGF‐1) is one of the important factors for growth, milk production and reproductive functions and mainly released from the liver in response to growth hormone (GH) via GH receptor (GHR) in cattle. Recently, some single nucleotide polymorphisms (SNPs) were identified in the bovine GHR gene. Some GHR‐SNPs were shown to be related to plasma IGF‐1 concentration in cattle. Hence, the capacity to IGF‐1 production in the liver might be affected by GHR‐SNP and associated with performance in the future. This study examined whether GHR‐SNP is associated with IGF‐1 production in the liver of pre‐pubertal heifers. In 71 Holstein calves, blood samples for genomic DNA extraction were obtained immediately after birth. To genotype the GHR‐SNPs in the promoter region, polymerase chain reaction (PCR) products were digested with restriction enzyme NsiI (cutting sites: AA, AG and GG). All heifers at 4 months of age were intramuscularly injected with 0.4 mg oestradiol benzoate. Blood samples were obtained from the jugular vein just before (0 h) and 24 h after injection. The number of AA, AG and GG at the NsiI site was 0, 17 and 54 respectively. In AG and GG, plasma GH concentrations were higher pre‐injection than 24 h post‐injection (p < 0.01). Moreover, plasma GH concentrations in AG post‐injection were higher than in GG (p < 0.05). In contrast, the GG genotype exhibited higher plasma IGF‐1 concentrations in pre‐injection than post‐injection (p < 0.01), although oestradiol did not change IGF‐1 concentration in the AG genotype. We conclude that the GG polymorphism in the promoter region of GHR is associated with a higher potential capacity of IGF‐1 production in the liver of cattle.     

Epistatic effects between pairs of the growth hormone secretagogue receptor 1a,growth hormone,growth hormone receptor,non‐SMC condensin I complex,subunit G and stearoyl‐CoA desaturase genes on carcass,price‐related and fatty acid composition traits in Japanese Black cattle

Growth hormone secretagogue receptor 1a (GHSR1a), growth hormone (GH), growth hormone receptor (GHR), non‐SMC condensin I complex, subunit G (NCAPG) and stearoyl‐CoA desaturase (SCD), are known to play important roles in growth and lipid metabolisms. Single and epistatic effects of the five genes on carcass, price‐related and fatty acid (FA) composition traits were analyzed in a commercial Japanese Black cattle population of Ibaraki Prefecture. A total of 650 steers and 116 heifers for carcass and price‐related traits, and 158 steers for FA composition traits were used in this study. Epistatic effects between pairs of the five genes were found in several traits. Alleles showing strain‐specific differences in the five genes had significant single and epistatic effects in some traits. The data suggest that a TG‐repeat polymorphism of the GHSR1a.5′UTR‐(TG)_n locus plays a central role in gene–gene epistatic interaction of FA composition traits in the adipose tissue of Japanese Black cattle.     

Development of discrimination markers between Japanese domestic and imported beef

In the meat industry, correct labeling of beef origins or breed is required to assure quality and safety. This paper describes the development of discrimination markers between Japanese domestic and imported beef from the United States (US) and Australia (AUS) based on a bovine 50K single nucleotide polymorphism (SNP) array using a total of 110 samples: Japanese Black (n = 50), Japanese Holstein (n = 50) and US cattle (n = 10). Genotyping information revealed 1081 SNPs as candidate markers that were polymorphic only in US cattle. The genotyping results by PCR – restriction length polymorphism in Japanese Black (n = 300) and Holstein cattle (n = 146) revealed that 11 SNPs had alleles specific to US cattle. Their allelic frequencies in US cattle (n = 108) ranged from 0.097 to 0.250 with an average of 0.178 and the combined identification probability of US cattle was 0.987. In addition, we also verified the applicability of these US‐specific markers to AUS cattle. Their allelic frequencies in AUS cattle (n = 280) ranged from 0.063 to 0.224 with an average of 0.137 and the combined identification probability of AUS cattle was 0.963. In conclusion, a set of these markers could be useful for discriminating between Japanese domestic and imported beef and would contribute to identify origins and prevent falsified labeling of beef.     

The T allele at the g.1471620G>T in the EDG1 gene associated with high marbling in Japanese Black cattle is at a low frequency in breeds not selected for marbling

Our previous study detected a single nucleotide polymorphism (SNP), g.1471620G > T , in the 5' flanking region of the endothelial differentiation sphingolipid G-protein-coupled receptor 1 ( EDG1 ) gene, which has been considered as a positional functional candidate for the gene responsible for marbling, and showed association of the g.1471620G > T SNP with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the g.1471620G > T SNP among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Short Horn, Holstein, and Brown Swiss breeds. The T allele at the g.1471620G > T SNP associated with high marbling was found at high frequency in Japanese Black breed that has been subjected to a strong selection for high marbling, while the allele was absent or at very low frequencies in the other breeds that have not been strongly selected for high marbling. Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequency of the T allele at the g.1471620G > T SNP in the EDG1 .     

A novel phenotype for Laron dwarfism in miniature Bos indicus cattle suggests that the expression of growth hormone receptor 1A in liver is required for normal growth

Mutations within the growth hormone receptor (GHR) gene that lead to an inactivated or truncated GHR protein cause abnormal growth and small adult size in a variety of species (Laron dwarfism). We studied a line of miniature Bos indicus cattle that have phenotypic (small mature size) and endocrine (increased blood growth hormone and decreased blood insulin-like growth factor-I concentrations) similarities to Laron dwarfs. Liver mRNA from miniature and control cattle was used to amplify a cDNA within the coding region of the GHR. The miniature cattle had GHR mRNA size (determined by Northern blot) and cDNA sequence that were similar to control cattle and, therefore, were unlike most Laron dwarf genotypes in which the GHR gene is mutated. Amounts of mRNA from liver as well as muscle (superficial neck and longissimus) were analyzed by ribonuclease protection assay for IGF-I, total GHR, GHR 1A (inducible, liver-specific GHR mRNA), and GHR 1B (constitutive GHR mRNA). Four control and five miniature bulls were tested. As expected, liver IGF-I mRNA was decreased in the miniature cattle (approximately 12% of control; P < 0.01). The amount of the total GHR as well as GHR 1A mRNA were also decreased in liver (17% and 19% of control, respectively; P < 0.01). Other GHR mRNA, including GHR 1B mRNA, were similar for miniature and control cattle. In muscle, there was a tendency (P < 0.10) for decreased IGF-I mRNA and increased GHR mRNA in miniature compared with control cattle. In summary, a novel phenotype for Laron dwarfism in Bos indicus cattle was associated with underexpression of GHR 1A mRNA, but not other GHR mRNA variants in liver. In addition to decreased GHR 1A mRNA, the miniature cattle had decreased liver IGF-I mRNA. Full expression of GHR 1A in liver, therefore, may be required for full liver IGF-I expression and normal growth.     

Identification of the core rumen bacterial taxa and their population dynamics during the fattening period in Japanese Black cattle

The rumen microbiota comprises a vast range of bacterial taxa, which may affect the production of high-quality meat in Japanese Black cattle. The aim of this study was to identify core rumen microbiota in rumen fluid samples collected from 74 Japanese Black cattle raised under different dietary conditions using 16S rRNA gene amplicon sequencing. In the rumen of fattening Japanese Black cattle, 10 bacterial taxa, showing >1% average relative abundance and >95% prevalence, irrespective of the dietary conditions and the fattening periods, were identified as the core rumen bacterial taxa, which accounted for approximately 80% of the rumen microbiota in Japanese Black cattle. Additionally, population dynamics of the core rumen bacterial taxa revealed two distinct patterns: Prevotella spp. and unclassified Bacteroidales decreased in the mid-fattening period, whereas unclassified Clostridiales, unclassified Ruminococcaceae, Ruminococcus spp., and unclassified Christensenellaceae increased during the same period. Therefore, the present study reports the wide distribution of the core rumen bacterial taxa in Japanese Black cattle, and the complementary nature of the population dynamics of these core taxa, which may ensure stable rumen fermentation during the fattening period.     

Gene expression of resistin and TNF-α in adipose tissue of Japanese Black steers and Holstein steers

The aim of the present study was to compare the expression of adipose tissue mRNA related to glucose metabolism between Japanese Black steers (n = 5) and Holstein steers (n = 5). We examined the expression of the resistin, tumor necrosis factor‐α (TNF‐α), glucose transporter 1 (GLUT1) and growth hormone receptor (GHR) genes using real‐time polymerase chain reaction of cDNA in adipose tissue. The cDNA sequence identified by 5′/3′‐rapid amplification of cDNA and the deduced amino acid sequence were highly conserved in human, porcine and murine resistin. Expression of resistin mRNA was significantly greater in Holstein steers than in Japanese Black steers. In contrast, expression of TNF‐α mRNA was slightly greater in Japanese Black steers. Expression of GHR mRNA was significantly greater in Japanese Black steers compared with the Holstein steers, although there was no significant difference in the expression of GLUT1 mRNA. However, the plasma non‐esterified fatty acid (NEFA), glucose, insulin and growth hormone concentrations did not differ between Japanese Black and Holstein steers. The present results show that there is a difference in the expression level of mRNA related to glucose metabolism between Japanese Black steers and Holstein steers.     

Conception rate of Holstein and Japanese Black cattle following embryo transfer in southwestern Japan

This study aimed to quantify and compare conception rates to embryo transfer (ET) of Holstein and Japanese Black cattle in southwestern Japan. A 10‐year retrospective epidemiological survey was conducted. The recipient numbers for Holstein and Japanese Black cattle was 621 and 527, respectively. Conception rates of Holstein and Japanese Black cattle during the study period were 45.4% and 42.3%, respectively. There was no significant difference between both breeds. However, a different trend of conception rate to ET in Holstein and Japanese Black cattle was observed. In Holstein cattle, conception rate in August to October was lower than in the other months and was significantly lower (p < .05) than in April. Particularly, conception rate in October of Holstein cattle was the lowest (31.0%). In Japanese Black cattle, conception rates in December (24.2%) and January (31.3%) were lower than in the other months. Conception rate of Japanese Black cattle declined as the temperature–humidity index (THI) decreased, exhibiting significantly lower levels in the ≤45 THI class than in any other THI class (p < .05). By contrast, in Holstein cattle, no relationship was observed between conception rate and THI on the day of ET. These observations suggest the importance of appropriate management that considers seasonal reactivity in each breed.     

A novel phenotype for Lardon dwarfism in miniature Bos indicus cattle suggests that the expression of growth hormone receptor 1A in liver is required for normal growth

Mutations within the growth hormone receptor (GHR) gene that lead to an inactivated or truncated GHR protein cause abnormal growth and small adult size in a variety of species (Laron dwarfism). We studied a line of miniature Bos indicus cattle that have phenotypic (small mature size) and endocrine (increased blood growth hormone and decreased blood insulin-like growth factor-I concentrations) similarities to Laron dwarfs. Liver mRNA from miniature and control cattle was used to amplify a cDNA within the coding region of the GHR. The miniature cattle had GHR mRNA size (determined by Northern blot) and cDNA sequence that were similar to control cattle and, therefore, were unlike most Laron dwarf genotypes in which the GHR gene is mutated. Amounts of mRNA from liver as well as muscle (superficial neck and longissimus) were analyzed by ribonuclease protection assay for IGF-I, total GHR, GHR 1A (inducible, liver-specific GHR mRNA), and GHR 1B (constitutive GHR mRNA). Four control and five miniature bulls were tested. As expected, liver IGF-I mRNA was decreased in the miniature cattle (approximately 12% of control; P < 0.01). The amount of the total GHR as well as GHR 1A mRNA were also decreased in liver (17% and 19% of control, respectively; P < 0.01). Other GHR mRNA, including GHR 1B mRNA, were similar for miniature and control cattle. In muscle, there was a tendency (P < 0.10) for decreased IGF-I mRNA and increased GHR mRNA in miniature compared with control cattle. In summary, a novel phenotype for Laron dwarfism in Bos indicus cattle was associated with underexpression of GHR 1A mRNA, but not other GHR mRNA variants in liver. In addition to decreased GHR 1A mRNA, the miniature cattle had decreased liver IGF-I mRNA. Full expression of GHR 1A in liver, therefore, may be required for full liver IGF-I expression and normal growth.     

Genetic association between GHSR1a 5′UTR‐microsatellite and nt‐7(C>A) loci and growth and carcass traits in Japanese Black cattle

We carried out a genetic association study between five nucleotide polymorphisms (5′UTR microsatellite ((TG)_n), nt‐7(C>A), L24V, DelR242 and Intron 1 microsatellite) of the GHSR1a gene and growth and carcass traits in 1285 steers sired by 117 Japanese Black bulls in a progeny testing program. We report herein, a significant association between the 5′UTR microsatellite and nt‐7(C>A) loci and growth and carcass traits. We also propose a translational hypothesis that the association is due to differences in the secondary structure of GHSR1b mRNA (the non‐spliced type with the 5′UTR microsatellite) among the GHSR1a gene haplotypes. Furthermore, we predicted the potential increase in profitability due to increased carcass weight in cow‐calf fattening enterprises through planned matings based on DNA testing of the 5′UTR microsatellite. Statistical analysis revealed that the 5′UTR microsatellite locus had a significant additive effect on carcass weight (CW) and average daily gain (ADG), but not on beef marbling score (BMS). One of the four major microsatellite alleles (19‐TG allele) with an allele frequency of 0.145, had a significantly (P < 0.0007) desirable effect on CW and ADG. We concluded that the 19‐TG allele could potentially be economically useful nucleotide markers for growth and carcass traits in Japanese Black cattle.     

Association of a single nucleotide polymorphism in ribosomal protein L27a gene with marbling in Japanese Black beef cattle

Marbling, defined by the amount and distribution of intramuscular fat, is an economically important trait of beef cattle in Japan. The c2‐11#2 expressed sequence tag (EST) has been previously shown to possess expression difference in musculus longissimus muscle between low‐marbled and high‐marbled steer groups, and to be located within genomic region of a quantitative trait locus for marbling. Thus, the ribosomal protein L27a (RPL27A) gene containing the c2‐11#2 EST sequence was considered as a positional candidate for the gene responsible for marbling. In the present study, a single nucleotide polymorphism (SNP) in the promoter region of the RPL27A, referred to as g.3109537C>T, was detected between the 2 steer groups. The SNP was associated with the predicted breeding value for beef marbling standard number by the analyses using Japanese Black beef cattle population. The effect of genotypes of the SNP on the predicted breeding value for subcutaneous fat thickness was not statistically significant. These findings suggest that the RPL27A SNP may be useful for effective marker‐assisted selection to increase the levels of marbling in Japanese Black beef cattle.     

The assessment of genetic diversity within and among the eight subpopulations of Japanese Black cattle using 52 microsatellite markers

Japanese Black cattle are at risk for genetic homogeneity due to intensive use of a few sires. Therefore, assessment of the actual genetic diversity of this breed is important for future breeding plans. In the present study, we investigated the genetic diversity within and among eight subpopulations of Japanese Black cattle using 52 microsatellite markers. The parameters for genetic diversity of Japanese Black cattle were comparable to those of other cattle breeds, suggesting that the relatively high genetic diversity of the breed. However, upon comparison among the eight subpopulations, the Hyogo subpopulation showed markedly low genetic diversity. The results of the pairwise F_ST values, phylogenetic network and structure analysis indicated that the Hyogo population has remarkably high level of genetic differentiation from other populations, while Yamagata, Niigata, Hiroshima and Kagawa populations have low levels of genetic differentiation. Furthermore, multidimensional scaling plots indicated that individuals in some subpopulations were separated from individuals in the other subpopulations. We conclude that while the overall genetic diversity of Japanese Black cattle is still maintained at a relatively high level, that of a particular subpopulation is significantly reduced, and therefore the effective population size of the breed needs to be controlled by correct mating strategies.     

Genome‐wide association studies identified variants for taurine concentration in Japanese Black beef

We performed genome‐wide association studies (GWAS) using the BovineSNP50 array to detect significant single nucleotide polymorphisms (SNPs) that may affect the concentration of 22 free amino acids and three peptides in Japanese Black beef cattle. A total of 574 Japanese Black cattle and 40,657 SNPs from the array were used for this study. Genome‐wide significant SNPs were detected for β‐alanine (three SNPs on chromosomes 22 and 29) and taurine (26 SNPs on chromosome 22). Importantly, the top two SNPs for taurine were highly significant (p = 6.2 × 10^?21), and the frequency of the increase‐concentration allele (Q) for taurine was found to be 0.73. The Q allele frequency of this population was similar to that of the other unrelated Japanese Black cattle, but different from that of the other breeds. In addition, the significant SNPs were not associated with carcass traits or fatty acid compositions. Interestingly, the top three of the four most significant SNPs for taurine were located near solute carrier family 6, member 6 (SLC6A6), which is a membrane transporter for taurine. We also found two associated variants in the 5′‐upstream region of SLC6A6; however, they were less significantly associated than the SNPs from the BovineSNP50 array.     

The g.841G>C SNP of FASN gene is associated with fatty acid composition in beef cattle

The objective of the current study is to evaluate the association between fatty acid composition and fatty acid synthase gene polymorphisms as responsible mutations. For this purpose, we selected seven previously reported single nucleotide polymorphisms (SNPs) in FASN gene, including one within promoter region (g.841G>C) and six non‐synonymous SNPs (g.8805C>T, g.13126C>T, g.15532A>C, g.16024A>G, g.16039C>T, g.17924A>G), and genotyped them in Japanese Black cattle. Genotyping results revealed that g.8805 C>T and g.17924 A>G were monomorphic loci. Genome‐wide association analysis including the other five SNPs revealed that only g.841G>C showed significant associations with the percentages of C14:0, C14:1, C16:1 and C18:1 at 5% genome‐wide significance level. In order to further evaluate the effect, we genotyped g.841G>C using additional three populations, including two Japanese Black populations and a Holstein cattle population. g.16024A>G was also genotyped and included in the analysis because it has been reported to be associated with fatty acid composition in Japanese Black cattle. In the result of analysis of variance, g.841G>C showed stronger effects on fatty acid percentage than those of g.16024A>G in all populations. These results suggested that g.841G>C would be a responsible mutation for fatty acid composition and contribute to production of high‐grade beef as a selection marker in beef cattle.     

Marker-assisted selection for forelimb-girdle muscular anomaly of Japanese Black cattle

Forelimb‐girdle muscular anomaly is a hereditary disorder of Japanese Black cattle characterized by tremors and astasia caused by hypoplasia of the forelimb‐girdle muscles. The locus responsible for this disorder has been mapped on a middle region of bovine chromosome 26. In this study, we applied marker‐assisted selection to identify the carriers of this disorder. Four microsatellite markers, DIK4440, BM4505, MOK2602 and IDVGA‐59, linked to the disorder locus were genotyped in 37 unaffected offspring of a carrier sire. Transmission of the mutant or wild‐type allele of the disorder locus of the sire to the 37 offspring was determined by examining the haplotypes of these markers. The results showed that nine and 18 of the 37 animals possessed the paternally transmitted mutant and wild‐type alleles, respectively, and therefore, the nine animals with the mutant allele were identified as carriers. We concluded that the marker‐assisted selection using these four markers can be applied for the identification of the carriers of forelimb‐girdle muscular anomaly of Japanese Black cattle.     

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.     

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.     

日本牛的遗传资源

本文扼要回顾了日本牛种资源的发展历史;介绍了日本牛的遗传资源状况和各主要牛种的特征特性;从日本牛的遗传可变性和遗传距离等方面分析了日本牛品种之间的遗传关系;并展望了日本黑毛和牛、日本褐牛、日本短角牛、日本无角牛、见岛牛、口子岛牛等日本牛种资源的发展前景。     

渤海黑牛种群现状及其保种利用问题

渤海黑牛是肉役兼用的优良品种,被列为我国受保护的地方良种。本文重点介绍了渤海黑牛种群现状及存在的问题,并对渤海黑牛的保种途径与利用措施以及渤海黑牛的杂交改良与开发利用进行了归纳总结,认为以我国优良地方黄牛品种为基础,选育具有中国特色的黄牛肉用新品种,是我国肉牛保种及提高生产效益的必由之路,这对其它地方品种的保护与利用具有一定的借鉴意义。