Caprine somatic cell nuclear transfer using in vivo matured oocytes collected by laparoscopic follicular aspiration

In vivo matured oocytes collected by laparoscopic follicular aspiration (LFA) from hormone treated female goats were used as recipient ooplasts for somatic cell nuclear transfer (SCNT). Japanese native (Shiba) goats were used as donor females and some donor females were used repeatedly (two or three times) at intervals of a few months. To induce synchronization of estrus, a sponge containing 0.5 g of progesterone was inserted into the vagina of each goat for 14 days. These animals were also treated with follicle stimulating hormone (FSH) in a series of 8 injections over 4 days. The first FSH injection was administered on the morning of day 9 of sponge insertion. On the morning of day 13, 50 µg of gonadotropin‐releasing hormone (GnRH) was injected into each animal. Twenty‐nine hours after GnRH injection, LFA was performed. After removal of cumulus cells, collected oocytes with the first polar body were selected and enucleated for nuclear transfer. Anterior pituitary cells isolated from an adult male Shiba goat were transfected with a DNA fragment containing the enhanced green flourescent protein gene and the puromycin resistance gene. A single donor cell was inserted into the perivitelline space of each enucleated oocyte and fusion was induced with one electric pulse of 20 V for 10 µs. The SCNT goat eggs were cultured in chemically defined medium at 38.5°C in 5% CO₂, 5% O₂, 90% N₂ for 9 days. By LFA, 396 oocytes were collected from a total of 30 females. After removal of cumulus cells, 64% of them extruded the first polar body. The percentage of SCNT goat eggs produced using in vivo matured oocytes which developed to the blastocyst stage (20–21%) was significantly higher (P < 0.05) than that produced with in vitro matured oocytes (3–8%).     

Novel plasmodesmata association of dehydrin-like proteins in cold- acclimated Red-osier dogwood (Cornus sericea)

Dehydrins are proteins associated with conditions affecting the water status of plant cells, such as drought, salinity, freezing and seed maturation. Although the function of dehydrins remains unknown, it is hypothesized that they stabilize membranes and macromolecules during cellular dehydration. Red-osier dogwood (Cornus sericea L.), an extremely freeze-tolerant woody plant, accumulates dehydrin-like proteins during cold acclimation and the presence of these proteins is correlated with increased freeze tolerance (Karlson 2001, Sarnighausen et al. 2002, Karlson et al. 2003). Our objective was to determine the location of dehydrins in cold-acclimated C. sericea stems in an effort to provide insight into their potential role in the freeze tolerance of this extremely cold hardy species. Abundant labeling was observed in the nucleus and cytoplasm of cold-acclimated C. sericea stem cells. In addition, labeling was observed in association with plasmodesmata of cold-acclimated vascular cambium cells. The unique association of dehydrin-like proteins with plasmodesmata has not been reported previously.     

Long-term growth analyses of Japanese cedar trees in a plantation: neighborhood competition and persistence of initial growth deviations

The individual growth of tree diameter at breast height (dbh) is analyzed in an even-aged plantation of Cryptomeria japonica from stand age of 45 to 94 years, to examine how the growth of individual trees has been affected by the changes in spacing resulting from thinning operations. At any age, a significant proportion (0.37–0.46) of the variation in dbh growth during a 5–11-year period was explained by dbh at the beginning of the period, probably due to greater leaf mass of larger trees. Next, either one-sided or two-sided competition was added to the model, by calculating the basal area (BA) of neighboring trees around each tree within a given radius or BA for trees having larger dbh than the focal tree within the radius. After preliminary analyses, a radius of 8 m was selected as the critical range for tree competition. Although both types of competition explained a significant proportion (0.09–0.43) of growth variation, one-sided competition was not significant at ages greater than 54 years. Based on the model at 45 years of age, the initial deviation of growth rate for each tree from the predicted rate was calculated and added to the models as a third variable. This raised the coefficient of determination up to 0.50–0.74. These findings have practical significance for forest plantation management, particularly for controlling the growth of standing trees via thinning, to produce high-quality timber in the future.     

Seasonal and yearly variations in light use and nitrogen use by seedlings of four deciduous broad-leaved tree species invading larch plantations

Several deciduous broad-leaved tree species, differing in leaf phenology, invade larch (Larix kaempferii (Lamb.) Carrière) plantations in Japan. The understory light environment of larch forests changes drastically between the leafy and leafless periods. To determine how the invading seedlings exploit the changing light environment, and if phenological differences reflect the light- and nitrogen-use traits of the seedlings, we measured leaf phenology, seasonal changes in light-saturated photosynthetic rate (P(sat)), leaf nitrogen (N) content (N(area)), chlorophyll/nitrogen ratio (Chl/N), specific leaf area (SLA) and N remobilization rate (NRMR) over 3 years. The mid-successional or gap-phase species, Magnolia hypoleuca Siebold & Zucc., had a short leafy period and high P(sat) and NRMR. In contrast, two late-successional tree species, Prunus ssiori Friedr. Schmidt, which undergoes leaf flush before larch, and Carpinus cordata Blume, which maintains green leaves until frost, both had low P(sat) and NRMR but exploited the opportunity for growth during the period when the larch canopy trees were leafless. Quercus mongolica Fisch. ex Ledeb. var. crispula (Blume) Ohashi, a mid-late-successional species that underwent leaf flush at the same time as the overstory larch, had values of photosynthetic parameters between those of the gap-phase and late-successional species. Among species, M. hypoleuca and Q. mongolica had higher photosynthetic rates and photosynthetic N-use efficiencies. In all species, the relationship between N(area) and P(sat) showed species-specific yearly fluctuations; however, there was no yearly fluctuation in the relationship between N(area) and P(sat) at CO2 saturation. Yearly fluctuations in the N(area)-P(sat) relationship appeared to be induced by changes in SLA and N-use characteristics, which in turn are affected by climatic variations.     

Effects of a severe typhoon on forest dynamics in a warm-temperate evergreen broad-leaved forest in southwestern Japan

The effects of a typhoon on forest dynamics and the response of major tree species were studied in a warm-temperate evergreen broad-leaved forest in southwestern Japan. The strongest typhoon on record (T9313) passed through this region in 1993. Return periods of typhoons over 30 ms⁻¹ in instantaneous wind velocity and T9313 were estimated to be 2.2 and 104.5 years, respectively. Approximately 10% of all stems suffered some damage from T9313 and annual stem mortality rose from 1.3 to 2.7%. The estimated period that the number of stems would fall below 10% of the initial was four years shorter with T9313-class typhoons than without them. Thus, the disturbance by T9313 was not catastrophic at the site although T9313 was an episodic typhoon. The short-term responses of major tree species to T9313 were classified into four types: 1) blunt-response type with little decrease and recruitment of stems in the DBH ≥ 5 cm class (Distylium racemosum), 2) retreat type with larger decrease than recruitment (e.g. Quercus acuta), 3) sharp-response type with a large decrease and much recruitment (e.g. Cinnamomum japonicum), and 4) advance type with less decrease than recruitment (Eurya japonica). Among the four regeneration types classified by previous studies (climax, light-demanding, subcanopy, and few-sapling), the climax and few-sapling types each showed a specific short-term response, the blunt-response and retreat types, respectively, that explains one aspect of the regeneration strategies of each type. On the other hand, the light-demanding or sub-canopy type showed multiple short-term responses, indicating that each regeneration type contains species with various regeneration strategies. In this paper, I analyzed data from the database that was compiled by the Aya Research Team.     

Effects of light and microsite conditions on tree size of 6-year-old Cryptomeria japonica planted in a group selection opening

We examined the extent to which direct and indirect measures of light and microsite conditions could explain variation in tree height and diameter at the base of 6-year-old Cryptomeria japonica trees planted in a group selection opening of about 0.32 ha on a steep slope at Shiiba, Miyazaki Prefecture, southern Japan. We first used the gap light index (GLI) and soil thickness (ST) as directly measured indices. For an indirect measure of light, we used a between-cohort competition index (BCI) estimated from the position and total height of residual trees. For indirect measures of microsite, we examined topographic indices (slope, plan and profile curvature, average slope gradient, and relative elevation) derived from digital elevation models (DEMs) with different resolutions ranging from 2 to 10 m. The multiple linear regression using GLI and ST explained about 45% of variation in tree size, while simple regression using only GLI explained about 35%. The contribution of ST was about half of GLI. The multiple regressions using BCI and the topographic indices did not explain any more variation than using BCI alone (R ² of about 0.26). We conclude that microsite conditions with shallower soil and steeper slope have negative effects on tree growth in group selection openings, although the relative importance is smaller than light conditions. More comprehensive studies considering several openings with more heterogeneous topography including different species are needed to generalize our growth prediction using the indirect measures, which are useful for practical forest management.     

Effects of heat treatment on brittleness of <Emphasis Type="Italic">Styrax tonkinensis</Emphasis> wood

A new approach is proposed for the evaluation of the brittleness of heat-treated Styrax tonkinensis wood. Heat treatment made wood more brittle when wood was heated at a higher temperature or for a longer time. The brittleness increased to four times that of the control when wood was heated at 200°C for 12 h. For treatment at 160°C, the increase in brittleness without any change in weight is thought to be possibly caused by the relocation of lignin molecules. At higher temperatures, loss of amorphous polysaccharides due to degradation is thought to become the main factor affecting brittleness. The crystallites that were newly formed after 2 h of treatment showed brittleness that was different from that of the inherent crystallites remaining after 12 h of heat treatment. This inherent crystalline cellulose possibly plays a role in brittleness. There is also the possibility of using color to predict the brittleness of heat-treated wood.     

Chemical analysis of the product in acid-catalyzed solvolysis of cellulose using polyethylene glycol and ethylene carbonate

Degradation and decomposition of cellulose were studied in an acid-catalyzed solvolysis treatment of biomass using polyethylene glycol (PEG) and ethylene carbonate (EC). The solvolysis reaction was followed by a typical reaction system of wood liquefaction that uses sulfuric acid catalyst at 140° or 150°C at atmospheric pressure. The methods of fractionation and chemical analysis of the degraded cellulose in the solvolyzed product are discussed. The solvolyzed product was separated into several fractions, and they were hydrolyzed to release glucose and levulinic acid to determine the quantity of glucosides and levulinates in the solvolysis product. The data clearly showed that the solvolysis reaction had the same mechanism when using PEG or EC. Degradation of cellulose leads to the formation of glucosides, which then decompose, resulting in a levulinic acid structure, and producing a water-insoluble fraction. The conversion rates of both glucosides and levulinates strongly depend on the reaction conditions of the solvolysis. In particular, EC promotes faster conversion of the reactions. The method discussed here is a chemical analytical technique for characterization of the products of wood liquefaction.     

Comparative functional anatomy of hindlimb muscles and bones with reference to aquatic adaptation of the sea otter

Although the sea otter (Enhydra lutris) is a complete aquatic species, spending its entire life in the ocean, it has been considered morphologically to be a semi-aquatic animal. This study aimed to clarify the unique hindlimb morphology and functional adaptations of E. lutris in comparison to other Mustelidae species. We compared muscle mass and bone measurements of five Mustelidae species: the sea otter, Eurasian river otter (Lutra lutra), American mink (Neovison vison), Japanese weasel (Mustela itatsi) and Siberian weasel (M. sibirica). In comparison with the other 4 species, E. lutris possessed significantly larger gluteus, popliteus and peroneus muscles, but smaller adductor and ischiopubic muscles. The popliteus muscle may act as a medial rotator of the crus, and the peroneus muscle may act as an abductor of the fifth toe and/or the pronator of the foot. The bundles of the gluteus superficialis muscle of E. lutris were fused with those of the tensor fasciae latae muscle and gluteofemoralis muscles, and they may play a role in femur abduction. These results suggest that E. lutris uses the abducted femur, medially rotated crus, eversion of the ankle and abducted fifth digit or extended interdigital web as a powerful propulsion generator. Therefore, we conclude that E. lutris is a complete aquatic animal, possessing differences in the proportions of the hindlimb muscles compared with those in other semi-aquatic and terrestrial mustelids.