Three-dimensional readout of flash x-ray images of living sperm in water by atomic-force microscopy

The imaging of living specimens in water by x-ray microscopy can be greatly enhanced with the use of an intense flash x-ray source and sophisticated technologies for reading x-ray images. A subnanosecond [corrected] x-ray pulse from a laser-produced plasma was used to record the x-ray image of living sea urchin sperm in an x-ray resist. The resist relief was visualized at high resolution by atomic-force microscopy. Internal structure of the sperm head was evident, and the carbon density in a flagellum was estimated from the relief height.     

Immunohistochemical Study of Endocrine Cells in the Gastrointestinal Tract of the Philippine Carabao (Bubalus bubalis)

The distribution and frequency of occurrence of endocrine cells in the gastrointestinal tract of the Philippine carabao (Bubalus bubalis) were studied by immunohistochemistry. Fourteen types of immunoreactive (IR) endocrine cells were revealed. Among the cell types, only chromogranin, serotonin, and bovine pancreatic polypeptide (BPP) were present in the entire gut, while the others showed restricted distribution: somatostatin, gastrin, and cholecystokinin in the abomasum and small intestine; methionine-enkephalin-Arg⁶-Gly⁷-Leu⁸, motilin, neurotensin, secretin, gastric inhibitory peptide, and substance P in the small intestine; peptide tyrosine tyrosine (PYY) in the large intestine: and glucagon in the whole intestinal tract. Most of the cell types showed peak density in the pyloric, duodenal, or rectal region. The highest cell type heterogeneity was observed in the duodenum. The distribution profile of the gut endocrine cells in the carabao is closely related to that in the Holstein cattle. Important findings include the occurrence of BPP-IR cells in the entire gut and the high frequency of PYY-IR cells in the large intestine.     

Development of a canine trypsin-like immunoreactivity assay system using monoclonal antibodies

The radioimmunoassay (RIA) for trypsin-like immunoreactivity (TLI) is one of the most sensitive and specific tests for detecting exocrine pancreatic insufficiency (EPI). An abnormally low serum TLI concentration (<2.5 ng/ml) indicates end-stage EPI. Although RIA methods can be used to detect canine serum TLI, these procedures are beyond the capabilities of most veterinary clinics and general laboratories. Using monoclonal antibodies (mAbs), we developed an enzyme-linked immunosorbent assay (ELISA) for canine TLI and incorporated it into an immunochromatographic test (ICT) for the diagnosis of EPI. The ELISA was linear over TLI concentrations of 1-100 ng/ml. Levels of intra-assay coefficients of variance (CVs) were 1.8-6.1%, inter-assay CVs were 5.1-9.8%, and the recovery of TLI added to two samples of canine serum ranged from 89 to 111 and 93 to 108%, respectively. Good correlation (correlation coefficient, 0.974) occurred between the TLI values obtained by the ELISA method and those by RIA from 56 clinical samples. Serum TLI values in clinically healthy dogs ranged from 7.8 to 29.2 ng/ml by ELISA, and those from dogs with EPI were 0.0-0.6 ng/ml. The values were 0.0-287.4 ng/ml for dogs with pancreatitis, and those from dogs with gastrointestinal disease were 5.5-58.9 ng/ml. The only statistically significant difference (P<0.01) occurred between the TLI level of healthy dogs and those with EPI. The ICT kit showed high reproducibility, and the TLI values yielding negative results differed significantly (P<0.01) from those returning positive results. The ICT kit yielded negative results (indicating EPI) from clinical serum samples with TLI concentrations of 0.0-4.1 ng/ml by ELISA. Both the ELISA and ICT kit are useful tools in the diagnosis of canine EPI.     

Stereological analysis of metanephros from domestic cat (Felis catus,Linnaeus 1798) embryos and fetus

Kidney diseases are the most common illness for cats with a prevalence seven times higher than in dogs. Metanephros is the last of three renal systems to be formed during the embryonic period, which then becomes the permanent kidney. The current work aimed to analyse the morphology and to quantify the structures present in the development of metanephros from domestic cat (Felis catus) embryos and foetuses. For this purpose, the evaluation of the biometric parameters of metanephros from cat embryos and foetuses was performed in addition to the quantification of renal corpuscles and volume of cortical and medullary layers by stereological analysis. The evaluated biometric parameters were weight, width, height, thickness and volume. The values of the measured biometric parameters increased throughout the gestational stages. The quantity of renal corpuscles gradually increased following the embryo-foetal development, mainly during the middle of the gestational stage. It was during this phase that morphologically, a complete corticomedullary division was observed. Although the difference in the quantity of renal corpuscles between the middle and the end of the gestational stages was not statistically significant, there was an increase in the volume of the medullary layer and a decrease in the volume of the cortical layer between these two stages. These findings suggest that the metanephros presents a progressive growth with the renal corpuscles following this development until the middle of the gestational stage. Starting from this phase, the differentiation of the corticomedullary layers can be seen with a significant increase in the medullary layer.     

Excessive application of farmyard manure reduces rice yield and enhances environmental pollution risk in paddy fields

The present study examined the effect of excessive application of farmyard manure (FM) on rice production and environmental pollution in paddy fields of Japan. A long-term field experiment was conducted during the period 1976–2006 to examine the trends of rice yield and yield components as affected by the excessive FM application (20 Mg ha^?1 year^?1 containing 110 kg N, 180 kg P₂O₅, and 320 kg K₂O). Rice growth, soil fertility, and surface water quality were also assessed in the final year (2006). The results obtained were compared with those of a conventional practice with recommended doses of inorganic fertilizer (IF), i.e. 85 kg N, 68 kg P₂O₅, and 53 kg K₂O ha^?1 year^?1, and an unfertilized control (CR). The excessive FM application resulted in a gradual decrease in grain yield, which was mostly explained by the reduction of grain fertility under the luxuriant rice growth. This reduction may have been due to the higher accumulation of soil nutrients such as N, P, and K. Moreover, the excessive FM application increased chemical oxygen demand, total P, and soluble K concentrations in the paddy surface water and their effluent loads compared to the conventional practice with the recommended IF application.     

One-step immunochromatography assay for detection of high-level canine serum trypsin-like immunoreactivity

We developed a one-step immunochromatography assay kit to measure high levels of canine trypsin-like immunoreactivity (cTLI) for bedside estimation of canine pancreatitis. The serum cTLI level can be determined within 10 min by visual comparison of color strengths in the test and reference zones. The serum cTLI levels determined by this method correlate well with canine TLI-ELISA and can be classified into 3 categories: cTLI levels higher than 60 ng/ml were considered positive; 20-60 ng/ml, weakly positive; and less than 20 ng/ml, negative. Twelve dogs suspected of pancreatitis were examined using this method; 4 dogs were positive, 2 were weakly positive, and 6 were negative. This test can detect a high level of serum cTLI and a positive result in the TLIH test will provide critical information for evaluation of pancreatitis in dogs.     

Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

The effects of twisting and type of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up in cattle

The effects of twisting and type (single- or double-lumen) of aspiration needle on the efficiency of transvaginal ultrasound-guided ovum pick-up (US-guided OPU) were investigated in cattle. The first study using slaughterhouse ovaries revealed that twisting of the needle during follicle aspiration improved the oocyte recovery rate without deleterious effects on the attachment of cumulus layers. Vacuum pressure affected the oocyte recovery and cumulus attachment, regardless of the needle type. The needle type did not affect the oocyte recovery or cumulus attachment with an optimized vacuum pressure. In the second study, US-guided OPU was performed in live cows using two types of needles with a vacuum pressure of 75 mmHg. The needle type did not affect the oocyte recovery or cumulus attachment of the recovered oocytes. The results revealed that twisting of the needle is effective in follicle aspiration, and suggested that a single-lumen needle is as useful as a double-lumen needle for US-guided OPU in cattle.