Evaluating the use of piezo manipulator,laser or their combination for blastocoel cavity puncture to improve cryopreservation outcomes of large equine embryos

The main difficulty of large equine embryo cryopreservation is the replacement of blastocoel fluid with cryoprotectant solution. The objective of this study was to improve the cryopreservation of large equine embryos with PMAP and/or LAP. Embryos were collected via the non-surgical transcervical procedure and divided into three groups based on their size (A ≤ 300 µm, 300 µm<B < 700 µm and C ≥ 700 µm). Six embryos 233–1360 µm in diameter were punctured via piezo manipulator and/or laser pulse before cryopreservation. All embryos were cryopreserved on a Cryotop®. Frozen-thawed embryos were cultured for 3h and transferred to the recipient mares. After one week, pregnancy was diagnosed by ultrasonography. Two of six embryos resulted in a positive pregnancy, the result of pregnancy in group A and B was positive, but in group C was negative, and further investigation is necessary for ≥700 µm embryos. The results showed laser-assisted puncture could be helpful to extract blastocoel fluid and replace it with cryoprotectant. This is the first positive pregnancy report in laser puncture-assisted frozen-thawed equine embryo (>300 µm). However, more research is required to find the best method for embryos ≥700 µm.     

Morphological reproductive characteristics of testes and fertilization capacity of cryopreserved sperm after the Fukushima accident in raccoon (Procyon lotor)

Since the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, we have established an archive system of livestock and wild animals from the surrounding ex-evacuation zone. Wildlife within the alert zone have been exposed to low-dose-rate (LDR) radiation for a long continuous time. In this study, we analysed the morphological characteristics of the testes and in vitro fertilization (IVF) capacity of cryopreserved sperm of racoons from the ex-evacuation zone of the FDNPP accident. The radioactivity of caesium-137 (¹³⁷Cs) was measured by gamma-ray spectrometry, and the measured radioactivity concentration was 300–6,630 Bq/kg in the Fukushima raccoons. Notably, normal spermatogenesis was observed in the seminiferous tubules of the testes, with the germinal epithelium composed of a spermatogenic cell lineage with no evident ultrastructural alterations; freeze-thawing sperm penetration ability was confirmed using the interspecific zona pellucida-free mouse oocytes IVF assays. This study revealed that the chronic and LDR radiation exposure associated with the FDNPP accident had no adverse effect on the reproductive characteristics and functions of male raccoons.     

Changes in element concentrations during Andosol formation on tephra in Japan

Andosol formation involves the rapid, abundant and in situ formation of non‐crystalline materials from tephra deposits. A large amount of humus complexed with Al also accumulates in the A horizons. As these materials are rich in Al or Fe compared to the parent tephra, the concentrations of the major and minor elements change significantly during Andosol formation. The objectives of this study were to examine how the rock type of the tephra and its weight loss during the formation process affect the changes in the element concentrations of Andosols. A total of 95 samples with different rock types from 18 pedons of Andosols in Japan were used to determine the total concentrations of 54 elements. Principal component analysis suggested that the degree of weathering and the rock types of parent tephra are the important factors in the variation of the element concentrations in Andosols. Three rock types, dacitic, andesitic and basaltic‐andesitic, were identified from the V and Zn contents of ferromagnetic minerals separated from the Andosol samples. Basaltic Andosols were identified from the abundant coloured volcanic glass and olivine in the sand fraction. Regarding concentrations of 41 elements, at least one group based on rock type was significantly different from one to three other groups at the P= 0.05 level. The content of oxalate‐extractable Si (Si_o), Al (Al_o) and Fe (Fe_o) was used as an index to show the extent of Andosol development. Relatively strong correlations between the element concentrations and Si_o, Al_o and Fe_o as well as other weathering indices were found in the andesitic samples. Among these elements, at least 27 (Be, Al, Ti, Fe, Y, Zr, Nb, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Hf, Ta, Tl, Pb, Th and U) were enriched in the Andosols and the increases in these concentrations were related to total weight loss due to soil formation processes.     

Regulative actions of dietary soy isoflavone on biological antioxidative system and lipid metabolism in rats

Male Sprague-Dawley rats, 4 weeks of age, were fed purified diets either with or without 0.2% soy isoflavones rich powder for 5 weeks to elucidate their direct functions such as antioxidative action and regulation of lipid metabolism. Dietary soy isoflavones decreased serum lipid peroxide level in rats. Levels of liver and serum alpha-tocopherol were higher in the rats fed isoflavone than in those fed isoflavones-free diet. Thus, dietary soy isoflavones exhibited mild antioxidative function in this animal experiment. Isoflavone metabolites from diet may act as scavengers of reactive oxygen species. Dietary soy isoflavones lowered hepatic 3-hydroxy-3-methylglutaryl CoA reductase activity, although liver cholesterol level was not modulated. However, the levels of serum cholesterol and triglyceride decreased by consumption of soy isoflavones. Therefore, dietary soy isoflavones may exhibit hypocholesterolemic and hypolipidemic functions. Moreover, dietary soy isoflavones lowered hepatic Delta6 desaturase activity. Reflecting this observation, Delta6 desaturation indices ((18:2(n = 6) + 18:3(n = 6))/20:4(n = 6)) of tissue lipids tended to be lower in rats fed isoflavones than in those fed isoflavones-free diet. This action may contribute to the prevention of inflammatory response by imbalance of eicosanoids. These observations suggest that the positive intake of soy isoflavones may reduce the risk of some cardiovasucular diseases through their radical scavenging function and hypocholesterolemic action.     

Simple and rapid determination of thiabendazole,imazalil, and o-phenylphenol in citrus fruit using flow-injection electrospray ionization tandem mass spectrometry

A simple and rapid analytical method for thiabendazole (TBZ), imazalil (IMA), and o-phenylphenol (OPP) in citrus fruit has been developed by using flow-injection electrospray ionization tandem mass spectrometry for the first time. The method involves the combined use of stable isotopically labeled internal standards (thiabendazole-(13)C(6), imazalil-d(5), and p-phenylphenol-d(9)) and a multiple reaction monitoring technique. The average recoveries for the fungicides at the tolerance levels (TBZ and OPP, 10 mg/kg; IMA, 5 mg/kg) ranged from 77 to 101%, with the coefficients of variation (CVs) ranging from 0.7 to 4.2% (n = 5). At half the tolerance levels (TBZ and OPP, 5 mg/kg; IMA, 2.5 mg/kg), the average recoveries ranged from 62 to 112%, with the CVs ranging from 0.7 to 8.4% (n = 5). The CVs of the average recoveries, obtained from lemon samples fortified with three fungicides at the tolerance levels, obtained on three different days over two weeks, ranged within 2%. The analysis time, including sample preparation and determination, is only 15 min.     

Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation

Phospholipase D (PLD)-mediated transphosphatidylation of squid skin lecithin with L-serine was examined to prepare docosahexaenoic acid-containing phosphatidylserine (DHA-PS). When a biphasic system with organic solvent and 0.2 M acetate buffer (pH 5.5) was used, PS synthesis was significantly affected by the amount of 3.4 M L-serine-containing acetate buffer. L-Serine concentration in the acetate buffer and choice of organic solvent were also crucial. In a typical reaction with 0.8 unit of PLD (Streptomyces sp.), 2.5 mL of ethyl acetate substrate solution containing 30 mg of squid skin lecithin in combination with 3 mL of 3.4 M L-serine-containing 0.2 M acetate buffer (pH 5.5), PS content in the recovered phospholipid fraction increased to 43.1% after 24 h. DHA composed 37.6% of fatty acids in the converted PS. This was the same DHA level as in the substrate. Phosphatidylcholine (squid skin PC, DHA 44.2%) in the squid skin lecithin was more effectively converted to PS than phosphatidylethanolamine.     

Mineral Composition of Clay Fractions and Oxygen Vacancies in Silt-Sized Quartz in Soils on the Ka-Etsu Plateau, Fukui, Central Japan—Possibility of Eolian Dust Brought from Northern Asia as Parent Material of Soils

Fine- to medium-textured soils are distributed on the Early Pliocene rocks and Pleistocene sandy sediments in the Ka-Etsu plateau. The Jingaoka (JIN) soil was classified into Aluandic Andosols by WRB, and Low-humic Non-allophanic Kuroboku soils by the Fourth Committee for Soil Classification and Nomenclature of the Japanese Society of Pedology, although it was similar morphologically to Yellow-Brown Forest soils. On the other hand, the Tomitsu (TMI) and Yamamuro (YAM) soils were classified into Hyperdystri-Chromic Cambisols by WRB, and Typic Yellow-Brown Forest soils by the Fourth Committee for Soil Classification and Nomenclature based on the morphological, physical and chemical properties. These soils were influenced considerably by the Akahoya tephra, especially the JIN soil. Based on the results of clay-mineralogical analysis and the measurement of the ESR-signal intensity associated with the oxygen vacancies in fine quartz, a large part of parent materials in the soils distributed on the Ka-Etsu plateau was derived from the eolian dust which was brought with the NW winter monsoon from the Precambrian sediments in the northern part of the Asian continent during MIS 2 (24–11 thousand years ago) at the Last Glacial Maximum. The parent materials of the fine-textured JIN soil located on the highest terrace near the rocky coastline were mostly composed of eolian dust, although the influenced of the Akahoya tephra was recognized. The parent materials of the TMI soil were admixed with a small amount of autochthonous materials from the sandy coast located windward. In the YAM soil which was located on the side of a hilly area distant from the coastline, the ESR-signal intensity in fine quartz was considerably low, and the admixture of autochthonous materials was also considered since the texture of this soil was coarser than that of the JIN and TMI soils.     

Proton Budget for a Japanese Cedar Forest Ecosystem

The proton budget for a Japanese cedar (Cryptomeria japonica) forest in Gunma Prefecture, Japan, was studied by estimating biogeochemical fluxes. The proton budgets were estimated for three individual compartments of the ecosystem: vegetation canopy, and the upper (O horizon + 0–10 cm) and lower (10–100 cm) soil layers. The dominant proton sources in the compartments were atmospheric deposition (1.2 kmol ha^?1 yr^?1), nitrification (5.1 kmol, ha^?1 yr^?1) and base-cation uptake by vegetation (8.0 kmol, ha^?1 yr^?1) respectively. These proton sources were neutralized almost completely within the individual compartments mainly by base-cation release from the canopy or the soil. The sum of internal proton sources was five times as large as that of external ones. Nitrogen input from the atmosphere was 2.2 kmol ha^?1 yr^?1, whereas its output from the lower soil layer was 3.9 kmol ha^?1 yr^?1, indicating that a net loss of nitrogen occurred in the ecosystem. However, this did not cause the acidification of soil leachates because of a sufficient release rate of base cations from the soil.