In vitro development of bovine nuclear transfer embryos reconstructed with mammary gland epithelial cells at different passages

In the present study, the effect of passage of nuclear donor cells on the in vitro development of nuclear transfer (NT) embryos was investigated using colostrum‐derived mammary gland epithelial (MGE) cells at different passages (3–30 passages) to find reliable passages for the efficiency of cloning. Development of NT embryos to the blastocyst stage was affected by the number of passages of MGE cells (P < 0.05). Nuclear transfer embryos reconstructed with MGE cells at 3–7 passages showed a significantly higher blastocyst development (31.3–48.5%) than those with the cells at 10–30 passages (2.5–12.5%, P < 0.05). No difference in the proportion of the MGE cells with normal diploid was observed among passage of 3, 15 and 30 (P > 0.05). The use of MGE cells at early passages for nuclear donor cells may be advantageous for the production of NT embryos.     

Uniquely stable 40 kDa subfragment-2 in carp myosin

Digestion of carp myofibrils at 30 degrees C in 0.5 M KCl medium with calcium ion generated unique 135 kDa heavy meromyosin (HMM). The HMM was not produced when digested at 10 degrees C. A further digestion of the 135 kD HMM isolated in the absence of calcium ion generated uniquely short subfragment-2 (S-2) with a size of 40 kDa (40 kDa S-2) together with subfragment-1 (S-1). The 40 kDa S-2 was identified by N- and C-end sequencing, and demonstrated to locate the amino end of the rod portion. The unfolding temperature for the 40 kDa S-2 was around 52 degrees C as studied by circular dichroism measurement. The same unfolding peak was also detected with the intact rod together with a large unfolding peak at around 36 degrees C coming from the rest of the rod portion, light meromyosin. The unfolding peak for the 40 kDa S-2 in myosin was a little lower (48 degrees C) than that in free form, suggesting the involvement of the head portion in the stability of the 40 kDa S-2 in the structure.     

A comparison of regression methods for estimating soil-atmosphere diffusion gas fluxes by a closed-chamber technique

Continuous changes in methane (CH₄) and carbon dioxide (CO₂) concentrations inside a closed chamber were measured on the forest floor at three sites: a deciduous forest and a coniferous forest in Hokkaido, Japan, and a birch forest in West Siberia, Russian Federation. Flux estimations by three types of regression methods, exponential, nonlinear, and linear, were examined using field-collected concentration data. The pattern of change with time of the gas concentration in the headspace differed, mainly according to site but also, to a lesser extent, according to the gas. This was a function of both the chamber height and surface soil property relating to soil gas diffusion and the gas concentration profile. Flux estimations did not differ statistically between the exponential and nonlinear methods for either gas at any site, because both of those regression methods were based on diffusion theory. However, the flux values estimated by linear regression were significantly different from those estimated by the other two methods for both CH₄ and CO₂ at the deciduous forest site and for CO₂ at the coniferous forest site. Shortening the chamber deployment period improved the linearity of the curve, but did not completely eliminate the error. Our results suggest that linear regression is not a good model of the change in headspace concentration with time.     

Development of monoclonal antibodies (MAbs) to feline interferon (fIFN)-γ as tools to evaluate cellular immune responses to feline infectious peritonitis virus (FIPV)

Feline infectious peritonitis virus (FIPV) can cause a lethal disease in cats, feline infectious peritonitis (FIP). The antibody-dependent enhancement (ADE) of FIPV infection has been recognised in experimentally infected cats, and cellular immunity is considered to play an important role in preventing the onset of FIP. To evaluate the importance of cellular immunity for FIPV infection, monoclonal antibodies (MAbs) against feline interferon (fIFN)-γ were first created to establish fIFN-γ detection systems using the MAbs. Six anti-fIFN-γ MAbs were created. Then, the difference in epitope which those MAbs recognise was demonstrated by competitive enzyme-linked immunosorbent assay (ELISA) and IFN-γ neutralisation tests. Detection systems for fIFN-γ (sandwich ELISA, ELISpot assay, and two-colour flow cytometry) were established using anti-fIFN-γ MAbs that recognise different epitopes. In all tests, fIFN-γ production from peripheral blood mononuclear cells (PBMCs) obtained from cats experimentally infected with an FIPV isolate that did not develop the disease was significantly increased by heat-inactivated FIPV stimulation in comparison with medium alone. Especially, CD8(+)fIFN-γ(+) cells, but not CD4(+)fIFN-γ(+) cells, were increased. In contrast, fIFN-γ production from PBMCs isolated from cats that had developed FIP and specific pathogen-free (SPF) cats was not increased by heat-inactivated FIPV stimulation. These results suggest that cellular immunity plays an important role in preventing the development of FIP. Measurement of fIFN-γ production with the anti-fIFN-γ MAbs created in this study appeared to be useful in evaluating cellular immunity in cats.     

Phenolics composition and antioxidant activity of sweet basil (Ocimum basilicum L.)

The antioxidant activity of a methanolic extract of Ocimum basilicum L. (sweet basil) was examined using different in vitro assay model systems. The crude extract was fractionated on a Sephadex LH-20 column, and six fractions were identified. The DPPH scavenging assay system and the oxidation of the soy phosphotidylcholin liposome model system were used to evaluate the antioxidant activity of each fraction. Fraction IV showed the strongest activity followed by fractions V and VI. Phenolic compounds responsible for the antioxidative activity of the fractions were characterized by atmospheric pressure chemical ionization liquid chromatography-mass spectrometry. The major antioxidant compound in fraction IV was confirmed as rosmarinic acid by (1)H NMR and characteristic fragmentations in the mass spectrum. Moreover, the native of antioxidant activity of rosmarinic acid in the liposome system was examined. The results showed that one rosmarinic acid can capture 1.52 radicals, and furthermore, the existence of a synergistic effect between alpha-tocopherol and rosmarinic acid was revealed.     

Effects of Rice Flour Blends on Bread Texture and Staling

The effects of blending rice flour with wheat flour on bread texture and staling were investigated with three rice varieties with different amylose contents. For the texture analysis of bread crumb, the compression test, puncture test, and tensile test were performed. A flour blend containing rice flour suppressed the recovery of the crumb after compression. For the puncture test, blends with rice flour increased the distance to penetrate the crumb. The rupture strain measured with the puncture test decreased with staling of the bread crumb for all samples tested. The fresh bread crumb sample containing waxy rice flour had much greater extensibility in the tensile test than the other samples tested, but it was dramatically decreased after one day of storage. Endothermic enthalpies corresponding to retrograded amylopectin, which is part of the staling process, were also measured. The enthalpy of bread crumb from the blended flour was lower than that of wheat flour bread up to three days but was higher on day 4. A blend of rice flour thus reduced amylopectin retrogradation during early storage, but it was accelerated later. Bread blended with waxy rice flour showed the lowest enthalpy during storage.     

First demonstration of bovine herpesvirus 2 infection among cattle by neutralization test in Japan

Seroepidemiological surveys were performed on neutralizing antibody to bovine herpesvirus 2 (BoHV-2) among cattle in Japan. A total of 1,819 sera were collected from cattle in 27 prefectures from 1997 to 1998. Antibodies were detected in only 18 sera collected from 4 prefectures. However, the most prevalent areas of the infection were found in two islands located in the subtropical zone. Additional 353 sera were collected in three including these islands in 1999-2001.The antibody-positive rates in the farms in these islands ranged from 10% to 81.1%. It was confirmed that BoHV-2 was prevalent in these areas. However, the infection seemed to be latent, because no diseases have been noticed. This is the first report showing the presence of BoHV-2 infection among cattle in Japan.     

Comparison of estrus induction and subsequent fertility with two different intravaginal devices in ewes during the non-breeding season

Two experiments were conducted to compare the effect of estrus induction by controlled internal drug release (CIDR) and intravaginal cream containing 500 mg progesterone (P cream) in ewes during the non-breeding season. In the first experiment, twenty-four ewes were randomly grouped for two treatments with the different intravaginal devices for 12 days: Group A was the CIDR group and Group B was the P cream group. Blood was collected from all treated ewes, and progesterone (P(4)), estradiol 17-beta (E(2)) and luteinizing hormone (LH) concentrations were measured by enzyme immunoassay. In the second experiment, the conception rates from natural mating, estrus-detected AI (inseminated 12 h after estrus detection), or fixed-time AI (inseminated 42 h after removal of an intravaginal device) in 127 ewes treated with CIDR or P cream were compared. In Experiment 1, the rate of estrus induction and the time of estrus onset after device removal were 91.7% and 36.3 +/- 15.7 h in Group A, and 100% and 35.0 +/- 12.6 h in Group B, respectively. There were no significant differences between the devices. The mean plasma P(4) concentration in Group B was significantly (P < 0.01) lower than Group A between day -9 and day -1 (Day 0: the day of device removal). However, no significant differences were found in the mean E(2) concentrations of the two groups after treatment. The mean time of estrus onset in ewes with an observed LH surge and the time of LH surge after treatment were 23.3 +/- 8.7 h and 30.3 +/- 5.0 h for Group A and 27.6 +/- 6.5 and 26.3 +/- 8.0 h for Group B, respectively, and there were no significant differences. However, a significant difference (P < 0.05) was found in the mean time from the time of estrus onset to LH surge between Group A (6.4 +/- 6.7 h) and Group B (-1.3 +/- 4.1 h). In Experiment 2, the conception rates for natural mating, estrus-detected AI, and fixed-time AI were 55.0, 29.4, and 25.0% for Group A and 40.7, 25.0, and 42.1% for Group B, respectively, and there were no significant differences. These results suggest that the effect of induction of estrus and ovulation and the rate of conception after treatment were comparable to CIDR even though the plasma P(4) concentration of the P cream method tended to be low during the insertion period.