Exploring indicators of genetic selection using the sniffer method to reduce methane emissions from Holstein cows.

OBJECTIVE: This study aimed to evaluate whether the methane (CH4) to carbon dioxide (CO2) ratio (CH4/CO2) and methane-related traits obtained by the sniffer method can be used as indicators for genetic selection of Holstein cows with lower CH4 emissions. METHODS: The sniffer method was used to simultaneously measure the concentrations of CH4 and CO2 during milking in each milking box of the automatic milking system to obtain CH4/CO2. Methane-related traits, which included CH4 emissions, CH4 per energy-corrected milk, methane conversion factor (MCF), and residual CH4, were calculated. First, we investigated the impact of the model with and without body weight (BW) on the lactation stage and parity for predicting methane-related traits using a first on-farm dataset (Farm 1; 400 records for 74 Holstein cows). Second, we estimated the genetic parameters for CH4/CO2 and methane-related traits using a second on-farm dataset (Farm 2; 520 records for 182 Holstein cows). Third, we compared the repeatability and environmental effects on these traits in both farm datasets. RESULTS: The data from Farm 1 revealed that MCF can be reliably evaluated during the lactation stage and parity, even when BW is excluded from the model. Farm 2 data revealed low heritability and moderate repeatability for CH4/CO2 (0.12 and 0.46, respectively) and MCF (0.13 and 0.38, respectively). In addition, the estimated genetic correlation of milk yield with CH4/CO2 was low (0.07) and that with MCF was moderate (-0.53). The on-farm data indicated that CH4/CO2 and MCF could be evaluated consistently during the lactation stage and parity with moderate repeatability on both farms. CONCLUSION: This study demonstrated the on-farm applicability of the sniffer method for selecting cows with low CH4 emissions.

Exploring the size of reference population for expected accuracy of genomic prediction using simulated and real data in Japanese Black cattle.

BACKGROUND: Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. RESULTS: A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900-3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1-0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29-0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000-11,000 animals, the accuracy of GEBV for carcass traits can range 0.73-0.79, which is comparable to estimated breeding value obtained in the progeny test. CONCLUSION: Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000-11,000 animals can be a sufficient size of reference population for genomic prediction.

Genotypes and allele frequencies of buried SNPs in a bovine single-nucleotide polymorphism array in Japanese Black cattle.

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding; thus, data derived from SNP arrays have accumulated on a large scale nationwide. Commercial SNP arrays contain a considerable number of unassigned SNPs on the chromosome/position on the genome; these SNPs are excluded in subsequent analyses. Notably, the position-unassigned SNPs, or "buried SNPs" include some of the markers associated with genetic disease. In this study, we identified the position of buried SNPs using the Basic Local Alignment Search Tool against the surrounding sequences and characterized the relationship between SNPs and genetic diseases in Online Mendelian Inheritance in Animals based on the genomic position. We determined the position of 285 buried SNPs on the genome and surveyed the genotype and allele frequencies of these SNPs in 5,955 individual Japanese Black cattle. Eleven SNPs associated with genetic disease, which contained five buried SNPs, were found in the population with the risk allele frequency ranging from 0.00008396 to 0.46. These results indicate that buried SNPs in the bovine SNP array can be utilized to identify associations with genetic disorders from large scale accumulated SNP genotype data in Japanese Black cattle.

Relationship between call rate per individual and genotyping accuracy of bovine single-nucleotide polymorphism array using deoxyribonucleic acid of various qualities.

Single nucleotide polymorphism (SNP) arrays are widely used for genetic and genomic analyses in cattle breeding. However, the relationship among sample genotyping efficiency (call rate per individual), accuracy of SNP genotypes, and DNA quality (integrity, concentration, and mixture of DNA, i.e., chimerism) remains unknown. We determined the effect of DNA quality on call rate per individual and accuracy of SNP genotypes using artificial DNA samples of various qualities. Integrity and concentration of DNA were less sensitive to call rate per individual and accuracy of genotyping in the SNP array. Chimerism strongly affected call rate per individual and accuracy of SNP genotypes. Artificial chimerism experiments showed that relative to unmixed DNA, the genotypic matching error (%) of mixed DNAs linearly increased with mix ratio, whereas the call rate per individual in some samples at 50% mix ratio was >0.95. However, individuals with higher chimerism were readily identified based on standard deviation of B-allele frequency (BAF) and BAF distribution across the genome from SNP array data. Thus, we effectively managed the balance by maximizing genotyping accuracy and minimizing the number of samples for re-genotyping by using quality control for combining call rate per individual with BAF.

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 ß-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.

Effect of two non-synonymous ecto-5'-nucleotidase variants on the genetic architecture of inosine 5'-monophosphate (IMP) and its degradation products in Japanese Black beef.

BACKGROUND: Umami is a Japanese term for the fifth basic taste and is an important sensory property of beef palatability. Inosine 5'-monophosphate (IMP) contributes to umami taste in beef. Thus, the overall change in concentration of IMP and its degradation products can potentially affect the beef palatability. In this study, we investigated the genetic architecture of IMP and its degradation products in Japanese Black beef. First, we performed genome-wide association study (GWAS), candidate gene analysis, and functional analysis to detect the causal variants that affect IMP, inosine, and hypoxanthine. Second, we evaluated the allele frequencies in the different breeds, the contribution of genetic variance, and the effect on other economical traits using the detected variants. RESULTS: A total of 574 Japanese Black cattle were genotyped using the Illumina BovineSNP50 BeadChip and were then used for GWAS. The results of GWAS showed that the genome-wide significant single nucleotide polymorphisms (SNPs) on BTA9 were detected for IMP, inosine, and hypoxanthine. The ecto-5'-nucleotidase (NT5E) gene, which encodes the enzyme NT5E for the extracellular degradation of IMP to inosine, was located near the significant region on BTA9. The results of candidate gene analysis and functional analysis showed that two non-synonymous SNPs (c.1318C > T and c.1475 T > A) in NT5E affected the amount of IMP and its degradation products in beef by regulating the enzymatic activity of NT5E. The Q haplotype showed a positive effect on IMP and a negative effect on the enzymatic activity of NT5E in IMP degradation. The two SNPs were under perfect linkage disequilibrium in five different breeds, and different haplotype frequencies were seen among breeds. The two SNPs contribute to about half of the total genetic variance in IMP, and the results of genetic relationship between IMP and its degradation products showed that NT5E affected the overall concentration balance of IMP and its degradation products. In addition, the SNPs in NT5E did not have an unfavorable effect on the other economical traits. CONCLUSION: Based on all the above findings taken together, two non-synonymous SNPs in NT5E would be useful for improving IMP and its degradation products by marker-assisted selection in Japanese Black cattle.

Genomic evaluation using SNP- and haplotype-based genomic relationship matrices in a closed line of Duroc pigs.

A simulation analysis and real phenotype analysis were performed to evaluate the impact of three different relationship matrices on heritability estimation and prediction accuracy in a closed-line breeding of Duroc pigs. The numerator relationship matrix (NRM), single nucleotide polymorphism (SNP)-based genomic relationship matrix (GRM) (GS ), and haplotype-based GRM (GH ) were applied in this study. We used PorcineSNP60 genotype array data (38 114 SNPs) of 831 Duroc pigs with four selection traits. In both heritability estimation and prediction accuracy, the accuracy depended on the number of animals with records. For heritability estimation, a large difference in the results among three relationship matrices was not shown, but the trend of the estimated heritabilities between GRMs, that is GS  < GH , was shown in this population. For the accuracy of prediction values in test animals, the accuracies of prediction values obtained by two GRMs were higher than that by the NRM in this population. The accuracies obtained by GRMs using animals with no records were lower than that by the NRM using animals with their performance records, but were close to that by the NRM using animals with full-sib testing records.

Genome-wide association study for carcass traits, fatty acid composition, chemical composition, sugar, and the effects of related candidate genes in Japanese Black cattle.

We performed a genome-wide association study (GWAS) and candidate gene analysis to: (i) evaluate the effectiveness of the GWAS in our small population by performing GWAS for carcass weight (CW) and fatty acid composition; (ii) detect novel candidate regions affecting non-CW carcass traits, chemical composition and sugar; and (iii) evaluate the association of the candidate genes previously detected in CW and fatty acid composition with other economically important traits. A total of 574 Japanese Black cattle and 40 657 Single nucleotide polymorphisms were used. In addition, candidate gene analyses were performed to evaluate the association of three CW-related genes and two fatty acid-related genes with carcass traits, fatty acid composition, chemical composition and sugar. The significant regions with the candidate genes were detected for CW and fatty acid composition, and these results showed that a significant region would be detectable despite the small sample size. The novel candidate regions were detected on BTA23 for crude protein and on BTA19 for fructose. CW-related genes associated with the rib-eye area and fatty acid composition were identified, and fatty acid-related genes had no relationship with other traits. Moreover, the favorable allele of CW-related genes had an unfavorable effect on fatty acid composition.

SNP- and haplotype-based genome-wide association studies for growth, carcass, and meat quality traits in a Duroc multigenerational population.

BACKGROUND: The aim of the present study was to compare the power of single nucleotide polymorphism (SNP)-based genome-wide association study (GWAS) and haplotype-based GWAS for quantitative trait loci (QTL) detection, and to detect novel candidate genes affecting economically important traits in a purebred Duroc population comprising seven-generation pedigree. First, we performed a simulation analysis using real genotype data of this population to compare the power (based on the null hypothesis) of the two methods. We then performed GWAS using both methods and real phenotype data comprising 52 traits, which included growth, carcass, and meat quality traits. RESULTS: In total, 836 animals were genotyped using the Illumina PorcineSNP60 BeadChip and 14 customized SNPs from regions of known candidate genes related to the traits of interest. The power of SNP-based GWAS was greater than that of haplotype-based GWAS in a simulation analysis. In real data analysis, a larger number of significant regions was obtained by SNP-based GWAS than by haplotype-based GWAS. For SNP-based GWAS, 23 genome-wide significant SNP regions were detected for 17 traits, and 120 genome-wide suggestive SNP regions were detected for 27 traits. For haplotype-based GWAS, 6 genome-wide significant SNP regions were detected for four traits, and 11 genome-wide suggestive SNP regions were detected for eight traits. All genome-wide significant SNP regions detected by haplotype-based GWAS were located in regions also detected by SNP-based GWAS. Four regions detected by SNP-based GWAS were significantly associated with multiple traits: on Sus scrofa chromosome (SSC) 1 at 304 Mb; and on SSC7 at 35-39 Mb, 41-42 Mb, and 103 Mb. The vertnin gene (VRTN) in particular, was located on SSC7 at 103 Mb and was significantly associated with vertebrae number and carcass lengths. Mapped QTL regions contain some candidate genes involved in skeletal formation (FUBP3; far upstream element binding protein 3) and fat deposition (METTL3; methyltransferase like 3). CONCLUSION: Our results show that a multigenerational pig population is useful for detecting QTL, which are typically segregated in a purebred population. In addition, a novel significant region could be detected by SNP-based GWAS as opposed to haplotype-based GWAS.

A genome-wide association study reveals a quantitative trait locus for days open on chromosome 2 in Japanese Black cattle.

Days open (DO), which is the interval from calving to conception, is an important trait related to reproductive performance in cattle. To identify quantitative trait loci for DO in Japanese Black cattle, we conducted a genome-wide association study with 33,303 single nucleotide polymorphisms (SNPs) using 459 animals with extreme DO values selected from a larger group of 15,488 animals. We identified a SNP on bovine chromosome 2 (BTA2) that was associated with DO. After imputation using phased haplotype data inferred from 586 812 SNPs of 1041 Japanese Black cattle, six SNPs associated with DO were located in an 8.5-kb region of high linkage disequilibrium on BTA2. These SNPs were located on the telomeric side at a distance of 177 kb from the parathyroid hormone 2 receptor (PTH2R) gene. The association was replicated in a sample of 1778 animals. In the replicated population, the frequency of the reduced-DO allele (Q) was 0.63, and it accounted for 1.72% of the total genetic variance. The effect of a Q-to-q allele substitution on DO was a decrease of 3.74 days. The results suggest that the Q allele could serve as a marker in Japanese Black cattle to select animals with superior DO performance.

Impact of QTL minor allele frequency on genomic evaluation using real genotype data and simulated phenotypes in Japanese Black cattle.

BACKGROUND: Genetic variance that is not captured by single nucleotide polymorphisms (SNPs) is due to imperfect linkage disequilibrium (LD) between SNPs and quantitative trait loci (QTLs), and the extent of LD between SNPs and QTLs depends on different minor allele frequencies (MAF) between them. To evaluate the impact of MAF of QTLs on genomic evaluation, we performed a simulation study using real cattle genotype data. METHODS: In total, 1368 Japanese Black cattle and 592,034 SNPs (Illumina BovineHD BeadChip) were used. We simulated phenotypes using real genotypes under different scenarios, varying the MAF categories, QTL heritability, number of QTLs, and distribution of QTL effect. After generating true breeding values and phenotypes, QTL heritability was estimated and the prediction accuracy of genomic estimated breeding value (GEBV) was assessed under different SNP densities, prediction models, and population size by a reference-test validation design. RESULTS: The extent of LD between SNPs and QTLs in this population was higher in the QTLs with high MAF than in those with low MAF. The effect of MAF of QTLs depended on the genetic architecture, evaluation strategy, and population size in genomic evaluation. In genetic architecture, genomic evaluation was affected by the MAF of QTLs combined with the QTL heritability and the distribution of QTL effect. The number of QTL was not affected on genomic evaluation if the number of QTL was more than 50. In the evaluation strategy, we showed that different SNP densities and prediction models affect the heritability estimation and genomic prediction and that this depends on the MAF of QTLs. In addition, accurate QTL heritability and GEBV were obtained using denser SNP information and the prediction model accounted for the SNPs with low and high MAFs. In population size, a large sample size is needed to increase the accuracy of GEBV. CONCLUSION: The MAF of QTL had an impact on heritability estimation and prediction accuracy. Most genetic variance can be captured using denser SNPs and the prediction model accounted for MAF, but a large sample size is needed to increase the accuracy of GEBV under all QTL MAF categories.

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle.

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.

Effect of restricted suckling on the superovulatory response and reproductive performance in postpartum Japanese black cows.

The reproductive performance of postpartum cows is affected by factors such as suckling and nutrition. We investigated the effect of a restricted suckling period on the superovulatory response and the fertility after flushing in postpartum Japanese Black cows. Forty-seven postpartum cows were used in this study. At 7 days postpartum, the cows were divided into 2 groups: (1) continuous access to calves from birth to weaning at 3 months postpartum (ad libitum suckling group; n=20); and (2) twice daily suckling to the calves penned adjacent to them (restricted suckling group; n=27). All cows were initiated a superstimulatory treatment with a controlled internal drug releasing device and follicle stimulating hormone at 40 days postpartum. Embryos were nonsurgically collected at 7 or 8 days after estrus. After uterine flushing, the cows were again used for reproduction. There were no significant differences between the ad libitum and restricted suckling groups in terms of the numbers of transferable (6.7 ± 5.4 versus 7.9 ± 7.0) and freezable embryos (5.5 ± 4.9 versus 6.2 ± 7.0). In contrast, the interval to the first estrus after flushing in the restricted suckling group was lower (P<0.05) than that in the ad libitum suckling group (8.9 ± 5.7 days versus 27.9 ± 24.2 days). These results suggest that restricted suckling in postpartum Japanese Black cows does not affect the superovulatory response and embryo quality; however, it improves their fertility after flushing.

Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET.

BACKGROUND: Interferon tau (IFNT), which is secreted into the uterine cavity during the maternal recognition period (MRP), is a key factor for establishment of pregnancy. The present study aims to clarify the relationship between the ability of a bovine conceptus to produce IFNT during the MRP and the conceptus's ability to establish pregnancy. METHODS: In the first experiment, IFNT (0, 500, or 1000 micrograms) was administered into the uterine horn ipsilateral to the CL 16 or 17 d after standing estrus, and mRNA levels of IFN-stimulated gene 15-kDa protein (ISG15) and Mx2 in peripheral blood mononuclear cells (PBMCs) were determined. In the second experiment, we investigated ISG15 mRNA expression in PBMCs during the MRP in cattle after either artificial insemination (AI) or embryo transfer (ET). RESULTS: Intrauterine administration of IFNT stimulated ISG15 and Mx2 gene expressions in PBMCs in cattle, and there was a positive correlation between the expressions of peripheral markers and the quantity of IFNT administered. In pregnant and normal interestrous interval (< 25 d) cattle (nIEI cattle), expression levels of the ISG15 gene showed similar patterns after AI and ET, and ISG15 mRNA expression was increased in pregnant cattle but unchanged in nIEI cattle. In contrast, ISG15 gene expression in extended interestrous interval (greater than or equal to 25 d) cattle (eIEI cattle) differed after ET compared with AI. In eIEI cattle after ET, ISG15 gene expression increased, such that the value on day 18 was intermediate between those of pregnant and nIEI cattle. In eIEI cattle after AI, ISG15 gene expression did not increase throughout the observation period. CONCLUSIONS: The results of the current study indicate that the quantity of conceptus-derived IFNT can be estimated by measuring ISG15 mRNA levels in PBMCs from cattle. Using this approach, we demonstrate that ISG15 gene expression during the MRP in eIEI cattle differed after ET compared with AI. In addition, the modest increase in ISG15 gene expression in eIEI cattle after ET suggests that late embryo losses were due to delayed or insufficient growth of the conceptus during the MRP in cattle.

Nucleotide polymorphisms and the 5'-UTR transcriptional analysis of the bovine growth hormone secretagogue receptor 1a (GHSR1a) gene.

Growth hormone secretagogue receptor 1a (GHSR1a) mediates the different actions of its endogenous ligand, ghrelin. Ghrelin-GHSR is involved in many important functions that include growth hormone secretion and food intake. We evaluated the haplotype variety and characterized the microsatellite ((TG)(n) , 5'-UTR) and nucleotide polymorphisms of the bovine GHSR1a gene. The nucleotide sequencing of this gene (∼6 kb) revealed 47 single nucleotide polymorphisms (SNPs), four indels and the microsatellite ((GTTT)(n) , Intron 1). The 19 haplotypes were constructed from all nucleotide viability patterns and were divided into three major groups. Four SNPs (L24V, nt456(G>A), D191N and nt667(C>T)) and DelR242 in Exon 1 and a haplotype block of approximately 2.2 kb (nt667(C>T) ∼ nt2884 (A>G)) were found in Bos taurus breeds. Breed differences in allele frequencies of the two microsatellites, nt-7(C>A), L24V, and DelR242 loci were found (P < 0.005). A DelR242 was found in the Japanese Shorthorn (frequency: ∼ 0.44), Japanese Brown, five European cattle breeds, the Philippine native cattle, but none detected in the Japanese Black or the Mishima island cattle. Additionally, 5'-rapid amplification of cDNA ends and RT-PCR analyses revealed that there were two different kinds of transcripts: spliced, without a microsatellite within 5'-UTR (GHSR1a); and non-spliced, with the microsatellite (GHSR1b).

Concentration of macrophage colony-stimulating factor (M-CSF) in bovine peripheral blood during pregnancy.

Macrophage colony-stimulating factor (M-CSF) is a hemopoietic cytokine with a primary role in placental physiology. Gene expression of M-CSF in the bovine endometrium shows a temporal upward trend during early and mid pregnancy. This study determined the plasma M-CSF levels during pregnancy using ELISA. In experiment 1, to investigate the relationship between the concentration of M-CSF in peripheral blood and pregnancy, the plasma M-CSF levels were determined in 125 pregnant and 21 non-pregnant Japanese Black cows. The pregnant animals were divided into nine groups based on the month of pregnancy. An ELISA for bovine M-CSF established previously was used according to the authors' instructions. In experiment 2, the plasma M-CSF level was determined to investigate the temporal changes in its concentration in the peripheral blood during pregnancy. In experiment 1, the plasma M-CSF level varied from month to month during pregnancy; the mean level in the first-month of pregnancy was significantly higher than those in the third and last months of pregnancy and non-pregnancy (P<0.05). In experiment 2, the plasma M-CSF level varied with the day of pregnancy (P<0.05). The mean level of plasma M-CSF decreased gradually until 6 weeks of pregnancy; it appeared to increase during weeks 7-9, then varied with several small peaks until 27 weeks of pregnancy and finally decreased gradually until parturition. These results suggest that the plasma M-CSF level may be related to changes in the uterus and placenta as pregnancy progresses.

Effect of total cholesterol, glucose and blood urea nitrogen on embryo quality in post-partum superovulated suckling Japanese Black cattle.

Aim: This study was conducted to examine the effect of blood metabolites on embryo quality in post-partum suckling Japanese Black cattle. Methods: Blood samples were taken from 23 cows 30 days before, at and 30 days after parturition. Cows were synchronized 40 or 41 days after calving (day 0) and divided into three groups: control (n = 6), gonadotropin-releasing hormone ([GnRH]n = 10) and estradiol benzoate ([EB]n = 7). All groups received a controlled internal drug release (CIDR) device intravaginally together with 2 mg EB i.m. on day 0 and superovulation was induced in all groups from days 5-7 with a gradually decreasing dose of follicle-stimulating hormone (FSH). Two milligrams of EB was given on day 8 and GnRH (0.1 mg) was given on day 9 of insertion of the CIDR in the EB and GnRH groups. Cows were inseminated twice after the onset of estrus and embryos were recovered 7-8 days after artificial insemination. Results: The number of corpus luteum detected by ultrasonography in the EB group was significantly higher (P < 0.05) than that in the GnRH group. The number and rate of transferable and freezable embryos did not differ significantly among the groups. Regardless of the treatments, the total cholesterol level from parturition until 30 days after parturition was significantly higher (P < 0.01) in the good category than in the poor category of cows. Conclusions: The number of transferable embryos produced by post-partum superovulated suckling Japanese Black cattle was affected by the level of total cholesterol from parturition until 30 days after parturition. Moreover, administration of EB in CIDR-treated cows increased the numbers of corpus luteum and yielded better rates of transferable and freezable embryos. (Reprod Med Biol 2008; 7: 55-62).

The bovine Mx1 gene: characterization of the gene structure, alternative splicing, and promoter region.

The Mx gene encodes an antiviral protein and is induced by type 1 interferons (IFNs). In this study, a new bovine Mx gene (designated Mx1B) was isolated from the endometrial cDNA library of the early pregnant cow. Although the Mx1B cDNA contained a single open reading frame (ORF) the same as the known Mx1, the 5' untranslated region (UTR) and 5' coding region of Mx1B were rather different from the corresponding regions of Mx1. Genomic structure analysis revealed that bovine Mx1B was an alternative splicing variant of Mx1 and had transcription regulatory sequences in the upstream region. RT-PCR and its sequencing identified another Mx1 splicing variant and demonstrated that these bovine Mx1 splicing variants were ubiquitously expressed in various tissues. Furthermore, it was found that all the bovine breeds investigated had identical splice sites of Mx1 and Mx1B. It is speculated that cattle have at least two functional Mx isoforms that might provide strong natural resistance to specific viruses.