Bos indicus type of growth hormone receptor gene is retained in Japanese Black cattle

The growth hormone receptor (GHR) gene is responsible for growth and carcass traits, and polymorphisms associated with the variation of meat production are thought to occur in the liver‐specific promoter of the GHR gene in cattle. The aim of this study was to analyse the structure of the liver‐specific promoter of GHR in Japanese Black cattle, as the relationship between GHR polymorphism and meat production is poorly understood in this breed. Typically in European cattle, the LINE‐1 element, a family of retrotransposons, is inserted in the liver‐specific promoter. However, a short GHR promoter without the LINE‐1 sequence was found in the Japanese Black breed as in Bos indicus cattle. The frequency of the short allele was approximately 60%. In addition, 24 of 29 Holstein/Japanese Black crosses carried the short allele from their sire. The present result suggests that the short allele for GHR may be a candidate marker for improving meat production of Japanese Black cattle.     

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.     

DGAT1, GH, GHR, PRL and PRLR polymorphism in water buffalo (Bubalus bubalis)

The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1‐exon8 K232A, GH‐intron3 MspI, GH‐exon5 AluI, GHR‐exon8 F279Y, PRL‐exon3 RsaI and PRLR‐exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili‐ravi, Murrah‐Nili‐Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR–Single‐strand conformation polymorphism and sequencing. Twenty‐eight inter‐specific single‐nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra‐specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1‐exon8, MspI⁺ restriction site(c nucleotide) and AluI⁺ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI^? restriction site at PRL‐exon3/exon4 and N allele genotype of S18N mutation at PRLR‐exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra‐specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL‐exon3/exon4 and 43rd bp (c/t) in PRLR‐exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances.     

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.     

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.     

The polymorphism in the β4-defensin gene and its association with production and somatic cell count in Holstein-Friesian cows

The aim of this study was to find a polymorphism of the bovine β4‐defensin gene and search for its association with milk yield and composition and with the somatic cell count in milk. The data were from the years 1999 to 2004 on 212 Holstein‐Friesian (HF) dairy cows, descended from 70 sires. Based on the sequence of the bovine β4‐defensin gene (GenBank no. AF008307 ) the primers were designed for the amplification of the 924‐bp or 393‐bp long fragments. The 924‐bp long fragment was sequenced and the sequence was compared with that available in the GenBank. Ten putative nucleotide sequence polymorphisms were found in the intron of the bovine β4‐defensin gene. One of them, a C→T transition at position 2239, that creates a new NlaIII (Hin1II) restriction site, was genotyped with polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) in a cohort of 212 HF cows. The CC genotype was the most common (72%). The heterozygous CT genotype was found in 26% of the genotyped cows and four cows (2%) were TT homozygotes. In order to determine the relationship between the polymorphism of the β4‐defensin gene and milk production traits a multi‐trait repeatability test‐day animal model was used. The Derivative‐free Multivariate analysis program was used for computation. The differences between estimates for genotypes were checked using Student's t‐test. The model included the animal genotype, year‐season of calving and parity as fixed effects and the animal additive genetic effect and permanent environmental effect of individual cows as well as dates of the tests as random effects. Significant associations were found between the RFLP‐NlaIII and milk fat, protein and lactose contents. Also, a significant effect was shown of the defensin genotype on the somatic cell count in the milk.     

Mitochondrial DNA analysis of Nepalese domestic dwarf cattle Lulu*

Dwarf Lulu cattle, the only Bos Taurus type of cattle in Nepal, are raised under severe environments in the mountainous zone of that country. In the present study, the body measurement traits, cytogenetic and molecular genetic characteristics of the Lulu cattle are investigated. Blood samples were collected from 31 animals in four villages (altitudes 2590–3550 m) in the southern part of Mustang. The Lulu cattle had a normal karyotype with 2n = 60, XY or XX. Only one male examined had a large submetacentric X‐chromosome and a small submetacentric taurine type Y‐chromosome. The mitochodrial DNA (mtDNA) genotypes were analyzed by PCR mediated restriction fragment length polymorphisms, displacement (D)‐loop region PCR mediated single strand conformation polymorphisms, and D‐loop region sequences. Many base substitutions were found in the D‐loop region, suggesting that the Lulu cattle originated from at least 10 maternal lines. Three types of mtDNA from these cattle were found, the Bos taurus type (n = 23), the Bos indicus type (n = 6), and the Bos grunniens type (n = 2). In the village at the lowest altitude, four of the five cows were of the Bos indicus type. These results indicated that mtDNA types of the Lulu cattle mostly belong to Bos taurus, but have been hybridized with Bos indicus cattle in lower‐elevation regions in their maternal lineage.     

Allele distributions of two novel SNPs within the sheep Cyp19 gene

Oestrogen is an important regulator of reproduction and growth. The key enzyme of oestrogen biosynthesis, aromatase cytochrome P450, is encoded by the Cyp19 gene. In order to generate genetic markers for the sheep Cyp19 gene, two novel single nucleotide polymorphisms (SNPs), one located in promoter 2 (P2), the other one in intron 9 (I9), were identified by a comparative sequencing approach. The allele distributions of both SNPs were investigated by means of Polymerase chain reaction‐restriction fragment length polymorphism analysis (PCR‐RFLP) in five economically relevant sheep breeds (British Milk Sheep, Carranzana, Latxa Black Face, Latxa White Face, Merino) and three ancient Hungarian breeds kept as gene reserves (Cikta, Racka, Tsigai). In British Milk Sheep, only the intronic SNP was present whereas in Merino, Cikta, Racka, Tsigai, Carranzana, Latxa Black Face and Latxa White Face, both SNPs could be found. This indicates that the newly identified SNPs can be used as markers for the Cyp19 locus in various sheep breeds.     

Identification of ovalbumin phenotypes of the Asian indigenous chicken populations using polymerase chain reaction-restriction fragment length polymorphism

Three electrophoretic variations (AA, BB and AB) of ovalbumin controlled by codominant alleles Ov^A and Ov^B have been observed in various chicken populations. We compared nucleotide sequences of the open reading frame between two alleles of ovalbumin gene. The difference between the two alleles was found as a non‐synonymous substitution of asparagine to aspartic acid as a result of AAT to GAT point mutation at position 8032–8034 in exon 8. We developed polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) protocol in combination with Mbo I restriction endnuclease mapping for the detection of this substitution. By the PCR‐RFLP the allelic frequency of the Ov^B was estimated to be within the range of 0.000–0.150 in 11 Asian indigenous chicken populations and 0.000 in four improved breeds used in the present study. Gene frequency, estimated by PCR‐RFLP in the present study, paralleled that obtained by protein polymorphisms of egg white. Thus, this study provides, for the first time, information of the occurrence of ovalbumin allele Ov^A and Ov^B in Asian indigenous chicken populations.     

Polymorphisms in the bovine hemoglobin‐beta gene provide evidence for gene‐flow between wild species of Bos (Bibos) and domestic cattle in Southeast Asia

The electrophoretic variation in bovine hemoglobin‐beta (HBB) is one of the most investigated genetic markers. The presence of a unique HBB variant, HBB^X, in Southeast Asian cattle has been reputed as a sign of gene‐flow from wild bovine species. In this study, we analyzed the DNA sequences of HBB genes in domestic and wild bovine species to verify this belief. Isoelectric focusing of HBB chain revealed that the HBB^X in domestic cattle had dimorphism and was separated into HBB^X1 and HBB^X2. The HBB^X1 had the same DNA sequence of the common HBB variant in gayal (Bos gaurus frontalis), while some of the HBB^X2 were identical with that of Cambodian banteng (Bos javanicus birmanicus). As a result, we confirmed that the bovine HBB variants can be a good indicator of introgression between wild and domestic cattle. The HBB^X1 was always predominant to HBB^X2 in the continental populations, suggesting that the gaur had contributed to the gene pool of domestic cattle in this region much more than the banteng. On the other hand, the mitochondrial DNA analysis could not detect gene‐flow from wild species. Autosomal markers that can trace the phylogeny between alleles are suitable for the assessment of bovine interspecific introgression.     

Differentiation of Twelve Actinobacillus pleuropneumoniae Serotypes by Outer Membrane Lipoprotein Gene‐based Restriction Fragment Length Polymorphism

Twelve Actinobacillus pleuropneumoniae serotypes were differentiated by restriction fragment length polymorphism (RFLP) of polymerase chain reaction (PCR)‐amplified fragments from the outer membrane lipoprotein (omlA) gene. All 12 reference serotypes and 80 field isolates produced the expected 950‐base pair (bp) fragment of the omlA gene by PCR. Combining the RFLP patterns obtained with SfaNI, Bst71I, AluI, NciI, nine distinct patterns were observed in the 12 serotype reference strains. The PCR‐based RFLP analysis of omlA genes allows differentiation among the 12 serotypes, with the exception of group 1 (serotypes 1, 9 and 11), and group 2 (serotypes 2 and 8). When the PCR products from the 70 field isolates were subjected to RFLP analysis, 68 showed the same RFLP patterns as their respective serotype reference strain. Two isolates that could not be typed had the same RFLP patterns as those of serotype 5. These results suggest that PCR‐based RFLP analysis of the omlA genes may be of value in differentiating among 12 A. pleuropneumoniae serotypes.     

Genome‐wide association study for feedlot average daily gain in Nellore cattle (Bos indicus)

The genome‐wide association study (GWAS) results are presented for average daily gain (ADG) in Nellore cattle. Phenotype of 720 male Bos indicus animals with information of ADG in feedlots and 354 147 single‐nucleotide polymorphisms (SNPs) obtained from a database added by information from Illumina Bovine HD (777 962 SNPs) and Illumina BovineSNP50 (54 609) by imputation were used. After quality control and imputation, 290 620 SNPs remained in the association analysis, using R package Genome‐wide Rapid Association using Mixed Model and Regression method GRAMMAR‐Gamma. A genomic region with six significant SNPs, at Bonferroni‐corrected significance, was found on chromosome 3. The most significant SNP (rs42518459, BTA3: 85849977, p = 9.49 × 10^?8) explained 5.62% of the phenotypic variance and had the allele substitution effect of ?0.269 kg/day. Important genes such as PDE4B, LEPR, CYP2J2 and FGGY are located near this region, which is overlapped by 12 quantitative trait locus (QTLs) described for several production traits. Other regions with markers with suggestive effects were identified in BTA6 and BTA10. This study showed regions with major effects on ADG in Bos indicus in feedlots. This information may be useful to increase the efficiency of selecting this trait and to understand the physiological processes involved in its regulation.     

Allele frequencies of gene polymorphisms related to economic traits in Bos taurus and Bos indicus cattle breeds

Allele frequencies of 10 representative polymorphisms for beef and milk traits were investigated for a total of 240 animals from Bos taurus and Bos indicus breeds, including two Japanese groups (Japanese Black and Japanese Brown), two East Asian groups (Korean and Mongolian), three European groups (Holstein, Angus and Hereford) and a Bos indicus group in South Asia (Myanmar, Laos and Cambodia). The Japanese Black revealed unique genetic construction in GH, FASN and SREBP‐1 and the other Asian populations show intermediate frequencies between European and Japanese populations. The Bos indicus group showed low favorable allele frequencies in most of the genes. The study showed the variability and distribution of 10 genes affecting economic traits among world representative cattle breeds. The genetic information would contribute to elucidating the genetic background for worldwide cattle breeds and the possibility of improvement using the markers.     

Kazakhstani native cattle reveal highly divergent mtDNA from Bos taurus and Bos indicus lineages with an absence of Bos indicus Y chromosome

Kazakhstan is the largest landlocked country and contains two important propagation routes for livestock from the Fertile Crescent to Asia. Therefore, genetic information about Kazakhstani cattle can be important for understanding the propagation history and the genetic admixture in Central Asian cattle. In the present study, we analyzed the complete mtDNA D‐loop sequence and SRY gene polymorphism in 122 Kazakhstani native cattle. The D‐loop sequences revealed 79 mitochondrial haplotypes, with the major haplogroups T and I. The Bos taurus subhaplogroups consisted of T (3.3%), T1 (2.5%), T2 (2.5%), and T4 (0.8%) in addition to the predominant subhaplogroup T3 (86.9%), and the Bos indicus subhaplogroup of I1 (4.1%). Subsequently, we investigated the paternal lineages of Bos taurus and Bos indicus, however, all Kazakhstani cattle were shown to have Y chromosome of Bos taurus origin. While highly divergent mtDNA subhaplogroups in Kazakhstani cattle could be due to the geographical proximity of Kazakhstan with the domestication center of the Fertile Crescent, the absence of Bos indicus Y chromosomes could be explained by a decoupling of the introgression dynamics of maternal and paternal lineages. This genetic information would contribute to understanding the genetic diversity and propagation history of cattle in Central Asia.     

The sequence of the bovine cysteine- and histidine-rich cytoplasmic gene: EST puzzle solving by comparative assembly and confirmation by genomic sequencing

A simple, yet comprehensive comparative mapping approach on how to mine the current GenBank database in order to facilitate the gene and quantitative trait locus (QTL) mapping in livestock species is described. Using this method, the bovine cysteine‐ and histidine‐rich cytoplasmic protein (CYHR1) gene was characterized. The cDNA sequence of the bovine CYHR1 was determined using public expressed sequence tag (EST) puzzle solving and its genomic organization was identified by sequencing. A BLAST search against the human genome sequences indicated that the bovine CYHR1 gene tentatively contains at least three exons and two introns, spanning approximately 4 kb in the bovine genome. A total of 149 and 155 cows with high or low estimated breeding values (EBVs) in protein yield were genotyped on a G/A polymorphism in intron 1 by polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) technique with digestion of restriction enzyme NlaIII. This substitution had no significant effects on protein yield in Canadian Holstein cattle, yet could be used as a marker for additional haplotype analysis.     

Comparison of Synthetic Oviductal Fluid and G1/G2 Medium under Low‐1 Oxygen Atmosphere on Embryo Production and Pregnancy Rates in Nelore (Bos indicus) Cattle

In this work, we evaluated whether embryo development and pregnancy rates would be affected by culturing bovine Bos indicus embryos in Synthetic Oviductal Fluid with amino acids (SOFaa) or G1/G2 sequential medium under a low‐oxygen atmosphere. Using Ovum Pick Up, we obtained 1,538 oocytes, divided into G1/G2 (n = 783) and SOFaa (n = 755). No difference was observed for blastocyst development among the groups (27.8% ± 14.6 and 34.9% ± 20.0 for G1/G2 and SOFaa respectively, p > 0.05). Transferring the embryos (n = 450) from both groups to recipients resulted in similar pregnancy rates for the G1/G2 (38.4% n = 78/203) compared to the SOFaa (39.7% n = 98/247). Our findings confirm that Bos indicus embryos cultured in SOFaa and G1/G2 under low‐oxygen atmosphere have similar in vitro (blastocyst rate) and in vivo (pregnancy rate) developmental capacity. However, embryos cultured in G1/G2 medium have higher cleavage than those cultured in SOFaa medium.