Determination of optimal conditions for parthenogenetic activation and subsequent development of rat oocytes in vitro

The present study was undertaken to determine optimal conditions for parthenogenetic activation and subsequent development of rat oocytes. Oocytes from immature Wistar-Imamichi (WI) and Sprague Dawley (SD) rats were activated by electrical stimulation in combination with 6-dimethylaminopurine (6-DMAP) to assess whether different rat strains display different responses to activation treatment. Since the cleavage rates of activated oocytes were significantly higher in WI than SD strain rats, WI rats were used for the subsequent experiments to determine the effects of post-hCG time, culture duration, different activation protocols (electrical stimulation with 6-DMAP or ionomycin with 6-DMAP) and osmolarity of the activation medium on the activation and subsequent development of WI rat oocytes. For oocytes activated by electrical stimulation combined with 6-DMAP, the percentages of oocytes that were activated and that developed to blastocysts were higher when oocytes were collected at 18-20 h than at any other time points after hCG injection (16, 22-24 h). Culturing for 2-6 h before activation treatment markedly decreased the percentage of activated oocytes that developed to beyond the four-cell stage. There were no differences in the percentages of oocytes with pronuclear formation and subsequent development to the two-cell and blastocyst stages between oocytes that were activated by electrical stimulation or ionomycin, both followed by 6-DMAP treatment. Activation of oocytes by ionomycin and 6-DMAP, both in low osmolarity media (246 mOsM), markedly increased the cleavage rates and percentages of high quality blastocysts (71%). The optimal conditions determined in the present study with simplified activation protocols and high efficiency of activation and subsequent development of WI rat oocytes will be helpful for further research involving nuclear transfer in the rat.     

Differentially methylated CpG sites related to fertility in Japanese Black bull spermatozoa: epigenetic biomarker candidates to predict sire conception rate

For semen suppliers, predicting the low fertility of service bull candidates before artificial insemination would help prevent economic loss; however, predicting bull fertility through in vitro assessment of semen is yet to be established. In the present study, we focused on the methylated CpG sites of sperm nuclear DNA and examined methylation levels to screen new biomarkers for predicting bull fertility. In frozen-thawed semen samples collected from Japanese Black bulls, for which the sire conception rate (SCR) was recorded, the methylation level of each CpG site was analyzed using human methylation microarray. According to regression analysis, 143 CpG sites related to SCR were significantly differentially methylated. Whole genome bisulfite sequence data were obtained from three semen samples and the differentially methylated regions (DMRs) that included the target CpG sites selected by human methylation microarray were confirmed. Using combined bisulfite restriction analysis, fertility-related methylation changes were detected in 10 DMRs. With the exception of one DMR, the methylation levels of these DMRs were significantly different between groups with high fertility (> 50%) and low fertility (< 40%). From multiple regression analysis of methylation levels and SCR, three DMRs were selected that could effectively predict bull fertility. We suggest that these fertility-related differences in spermatozoal methylation levels could be new epigenetic biomarkers for predicting bull fertility.     

Estimation of genetic parameters for carcass grading traits,image analysis traits,and monounsaturated fatty acids in Japanese Black cattle from Hyogo Prefecture

Genetic parameters for carcass grading traits, image analysis traits, and monounsaturated fatty acid (MUFA) percentages were estimated in 29,942 Japanese Black cattle from Hyogo Prefecture. The analyzed traits included five carcass grading traits, two image analysis traits, fat area ratio and fineness index, and two MUFA traits, one measured in intermuscular fat using near-infrared spectroscopy (NIRS) and the other in intramuscular fat using gas chromatography (GC). The heritability estimates of image analysis traits and MUFA were moderate to high, ranging from 0.395 to 0.740, and it was considered that they could be improved simultaneously with carcass grading traits because no severe genetic antagonism was observed. Although the heritability of the NIRS-based intermuscular MUFA was slightly lower than that of the GC-based intramuscular MUFA, the genetic correlation between the two methods was as high as 0.804. These results indicate that the NIRS method can be used as an alternative evaluation procedure to predict MUFA in intramuscular fat in the longissimus muscle.     

Receptor assay-guided isolation of anti-GABAergic insecticidal alkaloids from a fungal culture

The gamma-aminobutyric acid (GABA) receptor bears sites of action for insecticides. To discover GABA receptor-directed insecticides in natural products, fungal culture extracts were screened for their ability to inhibit specific binding of the radiolabeled noncompetitive antagonist [3H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane to housefly head membranes. The screening efforts led to the isolation of two alkaloids from Aspergillus terreus: PF1198A (alantrypinone) and PF1198B (serantrypinone), which had IC50 values of 0.34 and 2.1 microM, respectively, in this assay. These compounds were ca. 47-61-fold selective for housefly vs rat GABA receptors. Both compounds showed insecticidal activity against Myzus persicae in the range of 100-500 ppm. Binding assay-guided screening should provide significant opportunities for the identification of novel and selective insecticides.     

Glycopeptide derived from hen egg ovomucin has the ability to bind enterohemorrhagic Escherichia coli O157:H7

Ovomucin glycopeptide (OGP) was prepared by size exclusion chromatography after Pronase digestion of hen egg ovomucin, and the binding of OGP to foodborne pathogens (Bacillus cereus,Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, and Staphylococcus aureus) was investigaed. Binding assays with biotinylated bacteria as probes in microtiter plates showed that OGP bound to only E. coli O157:H7 among these foodborne pathogens. Periodate treatment markedly reduced the binding ability, indicating that E. coli O157:H7 bound to carbohydrate moieties of OGP. Lectin blot analysis with Maackia amurensis (MAA) and Sambucus nigra (SNA), which are specific for oligosaccharides containing sialic acid, revealed their binding sites in OGP were similar to the E. coli O157:H7 binding sites that were probed with biotinylated E. coli O157:H7 after Western blotting of OGP. Sialydase treatment of OGP abolished its ability to bind E. coli O157:H7, demonstrating that sialic acid played an important role in the binding. These results suggest that OGP has E. coli O157:H7-specific binding sites that consist of sialic acid. On the basis of these properties, OGP has the potential to be an ingredient with a protective effect against E. coli O157:H7 infection and to be a novel probe for the detection of E. coli O157:H7 in the food hygiene field.     

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by human cytochrome P450-dependent monooxygenase systems

Metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) by monooxygenase systems dependent on 12 forms of human cytochrome P450 (CYP) was examined with the recombinant yeast microsomes containing each of the human CYP. The metabolites of PCDDs were analyzed by HPLC and GC-MS. Remarkable metabolism by the multiple CYP forms was observed toward dibenzo-p-dioxin (DD) and mono-, di-, and trichloroDDs. The metabolism contained multiple reactions such as hydroxylation at an unsubstituted position, hydroxylation with migration of a chloride substituent, and hydroxylation with elimination of a chloride substituent. Although major CYPs in human liver such as CYP2C8, CYP2C9, and CYP3A4 showed no significant metabolism toward the PCDDs, CYP1A1 and CYP1A2 showed high catalytic activity toward DD and mono-, di-, and trichloroDDs. The kinetic parameters K(m)(app) and V(max) of the CYP1A1-dependent 8-hydroxylation activity of 2,3,7-trichloro-DD (2,3,7-triCDD) were estimated to be 0.30 microM and 51 (mol/min/mol of P450), respectively, suggesting that 2,3,7-triCDD was a good substrate for CYP1A1. However, none of the CYPs showed any detectable activity [<0.01 mol/min/mol of P450)] toward 2,3,7,8-tetraCDD. Substrate-induced absorption spectrum and inhibition studies indicated that CYP1A1 could bind 2,3,7,8-tetraCDD with considerably high affinity. It was strongly suggested that the long half-life (7.1 years) of 2,3,7,8-tetraCDD in humans was due to an extremely low activity of CYPs toward 2,3,7,8-tetraCDD in addition to its chemical stability.     

Detection of candidate polymorphisms around the QTL for fat area ratio to rib eye area on BTA7 using whole‐genome resequencing in Japanese Black cattle

In our previous study, we performed genome‐wide association study (GWAS) to identify the genomic region associated with Fat area ratio to rib eye area (FAR) and detected a candidate in BTA7 at 10–30 Mbp. The present study aims to comprehensively detect all polymorphisms in the candidate region using whole‐genome resequencing data. Based on whole‐genome resequencing of eight animals, we detected 127,090 polymorphisms within the region. Of these, 31,945 were located within the genes. We further narrowed the polymorphisms to 6,044 with more than five allele differences between the high and low FAR groups that were located within 179 genes. We subsequently investigated the functions of these genes and selected 170 polymorphisms in eight genes as possible candidate polymorphisms. We focused on SLC27A6 K81M as a putative candidate polymorphism. We genotyped the SNP in a Japanese Black population (n = 904) to investigate the effect on FAR. Analysis of variance revealed that SLC27A6 K81M had a lower p‐value (p = .0009) than the most significant SNP in GWAS (p = .0049). Although only SLC27A6 K81M was verified in the present study, subsequent verification of the remaining candidate genes and polymorphisms could lead to the identification of genes and polymorphisms responsible for FAR.     

The dietary effects of a fermented vegetable product on glutathione peroxidase activity and lipid peroxidation of Japanese flounder Paralichthys olivaceus

ABSTRACT:   To study the dietary effects of fermented vegetable product (FVP) on the protection against lipid peroxidation of various tissues in Japanese flounder, Paralichthys olivaceus . The fish were fed on experimental diets with or without FVP for 4 weeks. The glutathione concentrations in serum or liver had a tendency to increase on FVP-feeding fish. The FVP-feeding fish showed higher glutathione peroxidase (GPx) activity of liver than the control fish, and the GPx activity was increased by the administration of 6 mg/kg body weight/day FVP ( P  < 0.05). Conversely, fish fed on FVP containing diets exhibited significantly ( P  < 0.05) lower lipid peroxidants of serum and liver than the control fish. The FVP is suggested to suppress lipid peroxidation in the administrated fish, which led to enhancement of antioxidant effect against cultured fish.     

Age and growth of yellowstriped butterfish, <Emphasis Type="Italic">Labracoglossa argentiventris</Emphasis>, around Izu Oshima Island

ABSTRACT:   Age and growth of the yellowstriped butterfish, Labracoglossa argentiventris , around Izu Oshima Island were studied using a total of 1450 fish. Age was determined by counting the edge of the opaque zones as a ring mark on sectioned sagittal otoliths. Formation of the first ring was observed during spring or summer, corresponding to 1.5 years after hatching. Thereafter, one ring was formed each year in the same season as the previous year. The growth of the butterfish was rapid until 2 years of age. The maximum likelihood method was applied to the age and length data for estimating parameters in von Bertalanffy, Gompertz, and Logistic growth models. The selected model, based on the Akaike Information Criterion, was the von Bertalanffy growth model, which indicated differential asymptotic length and variance by sex.     

Simultaneous estimation of instantaneous mortality coefficients and rate of effective survivors to number of released fish using multiple sets of tagging experiments

ABSTRACT:   A method is proposed for estimating two instantaneous mortality coefficients, and rate of effective survivors to number of tagged fish released, using exact times at liberty from multiple sets of tagging experiments. Effective survivors are defined here by the product of survivors immediately after releasing and reporting rate of recaptures. A least-squares method was applied to estimate the parameters using statistical models expressing the numbers of effective survivors, which were calculated by a principle similar to virtual population analysis. In order to investigate the statistical performance of the method, simulation trials using operating models were carried out. The method was applied to data of tagging experiments for alfonsino Beryx splendens from 1957 to 1969, off Kanagawa Prefecture. In most of the results of simulation trials, the method yielded estimates of natural mortality coefficient of which expected values were within ± 12% of true values. Estimates of natural mortality coefficient, fishing coefficient and the rate of effective survivors for alfonsino are 0.57 (per year, 95% confidence limits [CL] 0.40–0.69), 0.094 (per year, 0.03–0.25) and 0.39 (0.12–1.00), respectively. Modifications for the generalization of the method and notes on its applications are discussed.