Japanese Society for Animal Reproduction: award for outstanding research 2002. The development and prevalence of the transfer technique for frozen-thawed embryos of Japanese black beef cattle in Tochigi Prefecture

The conditions of embryo transfer by the stepwise method, in which frozen-thawed embryos are transferred on day 7 (day 0=onset of estrus), were investigated with the aim of increasing pregnancy rates in frozen-thawed embryo transfer. The use of a vaginal speculum to prevent bacterial infection when passing an embryo transfer gun through the vagina yielded a pregnancy rate equal to or higher than that with application of a sheath cover to the transfer gun. Administration of a sedative, xylazine, to recipient cattle for preventing movement at the time of embryo transfer improved the pregnancy rate. The influence of the time from thawing of frozen embryos to transfer and of the transportation of the recipient by truck upon pregnancy rate was investigated. Embryo transfer within 60 minutes after aspiration into a straw or transportation of the bovine recipient, 1.5 hours each way before and after transfer, had no influence on pregnancy rate. Relations of the embryonic developmental stage and morphological quality after thawing of frozen embryos to pregnancy rate were investigated in recipients of nulliparous Holstein heifers. The pregnancy rate increased as the embryonic developmental stage advanced from compacted morula, early blastocyst, and blastocyst in that order. The pregnancy rate obtained with blastocyst stage embryos was significantly (P<0.05) higher than that with compacted morula stage embryos, and there was no significant difference in pregnancy rates between excellent morphological quality and good morphological quality for compacted morula stage embryos. When correlation of luteal function and pregnancy rate was investigated in bovine recipients, pregnancy rate showed a tendency to increase with increasing blood progesterone (P) concentration on the day before (on day 6 after estrus) and the day of embryo transfer. The pregnancy rate in bovine recipients, which showed a blood P concentration of > or =2.5 ng/ml on the day before embryo transfer, was significantly (P<0.05) higher than that in those with a blood P concentration of <2.5 ng/ml. Pregnancy rate showed a tendency to increase with decreasing blood estradiol-17beta (E2) concentration on the day of embryo transfer. Activation of luteal function by administration of human chorionic gonadotropin (hCG) in cycling cattle was investigated for its effect on increasing pregnancy rate in bovine recipients. A follicle coexisting with cyclic CL ovulated and induced CL formed after injection of hCG 1,500 IU 5 days after ovulation. The blood P concentration was significantly (P<0.05) higher in the administration group than in the control group, and the blood E2 concentration rapidly decreased, showing a lower concentration than in the control group. These results suggest the possibility that the pregnancy rate could be improved by administration of hCG. Pregnancy rate following intramuscular injection hCG 1,500 IU was comparatively investigated in parous Japanese Black beef cattle receiving frozen-thawed embryos 7 days after estrus. Pregnancy rate was 67.5% in the group in which hCG was administered on day 6 after estrus, and was significantly (P<0.05) higher than that in the control group (45.0%) and the group in which hCG was administered on day 1 after estrus (42.5%), revealing that hCG administration facilitated pregnancy. Transfer of frozen-thawed embryos in the blastocyst stage within 60 minutes after the aspiration into a straw, with a vaginal speculum after administration of xylazine is suggested as a way of improving pregnancy rate in bovine recipients with favorable luteal function and in those with luteal function activated by administration of hCG on the day before embryo transfer.     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

Effects of epidermal growth factor (EGF) on fetal islet B cells in vitro

ABSTRACT. It has been reported that when the rat fetus is treated with streptozotocin (STZ) in vivo, islet B cells are destroyed but later recover. To investigate the process of the recovery of B cells after in vitro treatment of the fetal pancreas with STZ and the role of epidermal growth factor (EGF) in the recovery of B cells, we measured the level of insulin released from the cultured fetal pancreas and examined it histologically. As a result, we immunohistologically confirmed the regeneration of B cells in the pancreas that had been cultured for 48 hr after destruction of islet B cells by STZ treatment. An immunohistologic study using proliferating cell nuclear antigen (PCNA) showed that without the addition of EGF, the cell division index was significantly higher in the STZ-treated group (STZ group) than in the untreated group (intact group), whereas with the addition of EGF, the cell division index increased in both groups, but EGF did not have a significant cell division-promoting effect on the pancreas in the STZ group. The addition of EGF caused a significant decrease in the concentration of insulin in culture medium in both groups. These results indicate that EGF has a cell growth-promoting effect on intact fetal pancreas in vitro but has the effect of inhibiting the release of insulin, and thus suggest that EGF does not trigger the regeneration of islet B cells.     

Dynamic excitation and static loading tests of glulam lattice floor

A wooden lattice floor with high stiffness and damping capacity has been developed to solve noise problems in wooden apartment houses. The lattice floor consisted of Douglas fir glulam beams with inserted steel plate joints and drift pins. To examine the structural performance of the floor, dynamic excitation and static loading tests were conducted on the full size floor. The first and second order resonance frequencies of the floor were 13.5Hz and 27.0Hz, respectively. These frequencies are similar to the peak frequency of a conventional wooden floor and the combined floor fabricated from glued laminated timber and iron. The maximum static load of the floor was 127kN. The apparent flexural rigidity was less than half the value of several floors studied in the past. However, it is considered that the stiffness is improved by constructing panels and this floor has almost equivalent performance. Relative deflection was not affected by the loading history.Part of this study was presented at the International Wood Engineering Conference, New Orleans, October 1996.     

The regeneration characteristics of Picea jezoensis and Abies sachalinensis on cut stumps in the sub-boreal forests of Hokkaido Tokyo University Forest

The regeneration characteristics of Picea jezoensis Carr. and Abies sachalinensis Masters on cut stumps in natural forest under selection cutting and natural regeneration were studied in a sub-boreal forest of the Hokkaido Tokyo University Forest. The following points were investigated: (1) what decay class of cut stumps are suitable for regeneration of the two conifer species; (2) what part, either a root collar or a cut surface, of a cut stump is suitable for regeneration; and (3) what percentages of cut stumps support conifer seedlings. The percentages of cut stumps that supported conifer seedlings increased as the decay class advanced, but slightly decreased in the latest stage of decay class, V. Both P. jezoensis and A. sachalinensis seedling densities per cut stump and per square meter of cut stumps increased as the decay class advanced. Some root collars in decay class IV supported A. sachalinensis seedlings, but few supported P. jezoensis. Densities of P. jezoensis per cut surface and per square meter of cut surface in decay class IV were higher than those of A. sachalinensis. On the other hand, densities of A. sachalinensis per root collar and per square meter of root collar in decay class IV were higher than those of P. jezoensis. Cut stumps in advanced decay classes are more suitable for regeneration of P. jezoensis and A. sachalinensis than those in early decay classes. Whereas a cut surface is more suitable for regeneration of P. jezoensis, a root collar is more suitable for regeneration of A. sachalinensis. Cut stumps are not sufficient to ensure enough conifer regeneration because not all conifer cut stumps in advanced decay classes support conifer seedlings.     

Structure and regeneration dynamics of dominant tree species along altitudinal gradient in a dry valley slopes of the Bhutan Himalaya

The aim of our study was to characterize the structure and regeneration dynamics of dominant tree species along altitudinal gradient in dry valley slopes of the Bhutan Himalaya. In the typical dry valley slopes of west-central Bhutan, we analyzed forest stratification, tree crown projection, and population structure from lower dry valley bottom (1520 m a.s.l.) to upper humid ridge top (3370 m a.s.l.) across five major forest types: i.e. (1) dry pine forest with Pinus roxburghii as a dominant species; (2) mixed broad-leaved forest with Quercus lanata (evergreen), Quercus griffithii, (deciduous), and Rhododendron arboreum (evergreen) as major canopy species; (3) evergreen broad-leaved forest dominated by Quercus semecarpifolia, Quercus glauca and Quercus oxyodon; (4) cool conifer forest dominated by Abies densa and Tsuga dumosa; (5) cold conifer forest with A. densa and Juniperus recurva as dominants. In general, regeneration pattern of major dominant species shifted from inverse-J (lower altitudes), to sporadic (mid-altitudes), and to uni-modal type (upper altitudes) corresponding to three regeneration trends: (1) invasive at the lower, warm, dry forest under relatively strong human disturbances; (2) stable/balanced at the mid-altitude, in a relatively stable, mature moist evergreen broad-leaved forest with gap regeneration; and (3) poor/low regeneration at the upper, cool, humid conifer forest with a continuously cattle-grazed understory. Overall, regeneration patterns were balanced in all forest types, however care should be taken to prevent excessive exploitation of dry pine forest of lower valley bottom, and to control cattle grazing at higher altitudes. Our findings can be incorporated into management plans for sustainable management and conservation of mountain forests.     

Effect of moisture content of members on mechanical properties of timber joints

To investigate the effect of moisture content (MC) of members on the mechanical properties of timber joints, bending tests of precut joints and shear tests of dowel-type joints were carried out using timbers of Japanese cedar (Cryptomeria japonica D. Don) with three moisture conditions: green, kiln-dried with a MC target of 15%, and over-kiln-dried with a MC target of 5%. For the bending test, timbers were processed with a precut processing machine into “koshikake-ari” (a kind of dovetail joint) and “koshikake-kama” (a kind of mortise and tenon joint). A pair of members was jointed together without mechanical fasteners. Bolts (diameter = 12 mm) and nails (diameter = 2.45 mm) were used as dowels in the shear test. Bolted joints were constructed with one bolt and two metal side plates. Two nails and two metal side plates were used for the nailed joint. For precut joints, no clear effect of MC was recognized on maximum moment and initial stiffness. The maximum strength of mechanical joints assembled with kiln-dried wood was changed by the degree of drying. Stiffness of the joints assembled with kiln-dried specimens was larger than that of the joints assembled with green specimens.Part of this study was presented at the 7th International IUFRO Wood Drying Conference, Tsukuba, July 2001     

Nutrient starvation affects expression of LC3 family at the feto-maternal interface during murine placentation

LC3 − the mammalian homolog of Atg8 − was found as autophagosome membrane binding protein in mammals and widely used as an autophagosomal marker. LC3A, B and C show different expression patterns in each tissue. The aim of this study was to reveal the differences of expression patterns among LC3 families in mouse placenta under normal condition and nutrient starving condition. LC3A and B were highly expressed in decidual cells. LC3A and B were increased in D14 compared with D12 and D16 in mouse placenta, while LC3C was decreased. Starvation induced increase in LC3B expression specifically. Immunohistochemistry showed different expression patterns among LC3A, B and C. LC3A expression in syncytiotrophoblast was vanished by starvation. The results of real time RT-PCR suggested differences between D12 and D16 in autophagic cascade induced by starvation. Taken together, this study suggests that autophagy could play a role in placental invasion system and that nutrient starvation affects LC3B expression.