Semen quality in a Giant Panda (Ailuropoda melanoleuca) in relation to estrus of a nearby resident female panda.

Semen quality was determined in a sexually mature male Giant Panda, electroejaculated 13 times during a 5-year interval, before, during and after estrus of a female Giant Panda housed nearby. Testis volume and plasma testosterone concentrations were also measured. Mean testis volumes were 1223.0 +/- 64.7(S.E.M.)cm3 (before estrus), 1213.2 +/- 218.2 cm3 (during estrus), and 1360.2+/-160.4 cm3 (after estrus). Compared to before and during estrus in the female, testis volume decreased 70 days after estrus and there was no projectile ejaculation. The mean semen volume and sperm count were 2.2+/-0.7 mL and 8.3 +/- 3.1 x 10(8) before estrus, 2.4 +/- 0.9 mL and 5.7 +/- 0.9 x 10(8) during estrus, and 1.3 +/- 0.3 mL and 8.1 +/- 1.7 x 10(8) after estrus, respectively. The semen volume, sperm count, and testis volume markedly differed from 90 days before estrus until 66 days after estrus, whereas no marked differences in sperm motility, sperm viability, and proportion of morphologically abnormal spermatozoa were observed. Plasma testosterone concentrations were elevated both before and during estrus (0.62 +/- 0.23 ng/mL and 0.95 ng/mL), but decreased substantially after estrus (0.20 +/- 0.0 ng/mL). We inferred that spermatogenesis was active in this male panda from approximately 3 months before estrus to 2 months after estrus in the adjacent female.

[Treatment and results of interstitial lung diseases in video assisted thoracoscopic lung biopsy].

In order to establish treatment of interstitial lung diseases in video assisted thoracoscopic lung biopsy, we retrospectively reviewed our experiences. The present study included 7 patients with a mean age of 46.4, range from 24 to 61, who were treated at our department from 1996 through 1999. They were 5 men and 2 women. The pathologic diagnosis was nonspecific interstitial pneumonia in 3 patients who responded to steroid therapy. Three other patients had usual interstitial pneumonia. One patient had lymphocytic interstitial pneumonia. No complications occurred. The results indicate that video assisted thoracoscopic lung biopsy is an effective and safe way to diagnose interstitial lung diseases.

Evaluations of p53 immunoreactivity, nucleolar organizer regions, and proliferating cell nuclear antigen in non-small cell lung carcinoma.

We examined p53 protein expression, proliferating cell nuclear antigen (PCNA), and argyrophilic nuclear organizer regions (AgNOR), in 102 patients with surgically-treated non-small cell lung cancer (NSCLC). p53 positive cases with DO-1 were defined when more than 10% of the tumor cell nuclei were stained. Mean AgNOR count and PCNA LI were 2.80 and 40.7 and there were no significant differences of AgNOR count and PCNA LI between p53 positive and negative cases. We assessed the relationship between the p53 immunoreactivity and various clinical or pathological parameters. p53 positive rate of stage III disease (46.3%) was significantly higher than that of stage II disease (28.6%). The p53 positive rate of squamous cell carcinoma (42.1%) tended to be higher than that of adenocarcinoma (33.9%). In the survival curves of patients with NSCLC according to the p53 immunoreactivity, there was no significant difference between p53 positive and negative cases. Eight potential prognostic parameters (p53 immunoreactivity, AgNOR count, PCNA LI, sex, age, year of operation, histology, and stage) were also estimated, using univariate and multivariate analysis. In univariate analysis, PCNA LI and AgNOR count, and stage were significantly related to shortened survival. In multivariate analysis, PCNA LI, Age, and stage were independently associated with shortened survival of NSCLC patients. PCNA staining may be more useful than p53 and AgNOR staining in assessing the aggressiveness of surgically-treated NSCLC, although the most useful clinical prognostic parameter should be achieved by the combined analysis of several prognostic indicators.

Determination of mirosamicin in animal tissues by high-performance liquid chromatography.

A simple and rapid method using high-performance liquid chromatography (HPLC) for the determination of mirosamicin in animal tissues has been developed. The drug was extracted with 0.3% metaphosphoric acid-methanol (7:3, v/v), and the extracts were cleaned on a Bond Elut SCX (500 mg) cartridge. The HPLC separation was performed on a Puresil 5C18 column (150 x 4.6 mm I.D.) with 0.05 M phosphate buffer (pH 2.5)-acetonitrile (70:30) as the mobile phase at a flow-rate of 0.5 ml/min; the drug was detected at 230 nm with 0.04 AUFS. The calibration graph was linear from 5 to 100 ng. The recoveries of microsamicin from various animal tissues fortified at 1.0 microgram/g were 83.7-88.6% with a relative standard deviation (R.S.D.) of 2.0-5.7%. The detection limit was 0.05 microgram/g.

Simultaneous determination of benofloxacin, danofloxacin, enrofloxacin and ofloxacin in chicken tissue by high-performance liquid chromatography.

A simple, rapid and reliable high-performance liquid chromatographic (HPLC) method for the simultaneous determination of residual fluoroquinolones (benofloxacin, danofloxacin, enrofloxacin and ofloxacin) in chicken has been developed. The drugs were extracted with 0.2% metaphosphoric acid-acetonitrile (7:3, v/v), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on a Wakosil II 5C18-HG column (150 x 4.6 mm I.D.) with 0.05 M phosphate buffer (pH 2.4)-acetonitrile (80:20, v/v) containing 2.5 mM 1-heptanesulfonic acid as the mobile phase. A fluorescence detector was used at an excitation wavelength of 295 nm and an emission wavelength of 455 nm. The calibration graphs were linear from 0.1 to 10 ng for danofloxacin and from 1 to 100 ng for benofloxacin, enrofloxacin and ofloxacin. The recoveries of the drugs from tissues fortified at a level of 0.2 microgram/g were 81.1-89.6%, and the detection limits were 0.01 microgram/g for ofloxacin, danofloxacin and enrofloxacin and 0.02 microgram/kg for benofloxacin. The time needed per sample was less than 60 min.

High-performance liquid chromatography of histamine and 1-methylhistamine with on-column fluorescence derivatization.

An on-column fluorometric derivatization method was developed for the determination of histamine and 1-methylhistamine (HMs) by high-performance liquid chromatography. The system for the derivatization consisted only of a commercially available single-plunger pump and a reversed-phase C18 column supported on synthetic polymer with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthalaldehyde as a derivatization reagent. It required no additional reaction system as for a post-column derivatization method. Injected HMs might be derivatized to a fluorophore on the inlet site of the high-performance liquid chromatographic column, followed by chromatography on the same column. Optimization of the on-column reaction conditions resulted in a simple and sensitive analytical method for the determination of HMs with excellent reproducibility and linearity of 0.05-5 micrograms/ml of both HMs. Application of this method to the determination of HMs in food samples resulted in a limit of quantification of 0.05 mg/100 g and in a greater than 95% overall mean recovery at a fortification of 0.1 mg/g of both HMs. This method was furthermore applicable to the determination of histamine released from rat peritoneal mast cells.

Simultaneous determination of residual synthetic antibacterials in fish by high-performance liquid chromatography.

A simple and rapid high-performance liquid chromatographic (HPLC) method for the simultaneous determination of sulphamonomethoxine (SMMX), sulphadimethoxine (SDMX), sulphisozole (SIZ), nalidixic acid (NA), oxolinic acid (OXA), piromidic acid (PMA), furazolidone (FZ) and sodium nifurstyrenate (NFSA) in cultured fish was developed. The drugs were extracted with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond Elut C18 clean-up procedure. The HPLC separation was carried out on an Inertsil ODS column (150 x 4.6 mm I.D.) using 5 mM aqueous oxalic acid-acetonitrile (55:45) as the mobile phase with detection at 265 nm (0.04 a.u.f.s.). The calibration graphs were rectilinear from 1 to 20 ng for OXA, from 2 to 50 ng for SMMX, SDMX, SIZ, NA, PMA and FZ and from 5 to 100 ng for NFSA. The recoveries of each drug added to fish were 65.0-89.5%. The detection limits were 0.02 micrograms/g for OXA, 0.05 micrograms/g for SMMX, SDMX, SIZ, NA, PMA and FZ and 0.1 micrograms/g for NFSA.

Identification and determination of sulphamethazine and N4-acetylsulphamethazine in meat by high-performance liquid chromatography with photodiode-array detection.

A simple and selective method for the determination of sulphamethazine (SMT) and its metabolite, N4-acetylsulphamethazine (N4-AcSMT), in meat by high-performance liquid chromatography (HPLC) with photodiode-array detection was developed. The drugs were extracted from meat with 0.2% metaphosphoric acid-methanol (6:4), followed by a Bond-Elut C18 clean-up procedure. The HPLC separation was carried out on a Supersphere RP-18e column (125 X 4.0 mm I.D.) using 0.05 M sodium dihydrogenphosphate (pH 4.5)-acetonitrile (8:2) as the mobile phase at a flow-rate of 0.5 ml/min, and monitored with a photodiode-array detector. The recoveries of SMT and N4-AcSMT from meat fortified at 0.5 micrograms/g were 90.1-93.3 and 93.0-94.4%, respectively, with coefficients of variation of 1.9-3.2 and 1.5-2.7%. The limits of detection were 0.02 micrograms/g for each drug. SMT was found in ten samples of imported meat (12.5%) at levels ranging from 0.05 to 1.05 micrograms/g.

Gas chromatographic determination of nicotinamide in meats and meat products as 3-cyanopyridine.

The determination of nicotinamide as 3-cyanopyridine after dehydration by heptafluorobutyric anhydride (HFB) was performed by gas-liquid chromatography (GLC) with flame ionization detection (GLC-FID) and a column of 5% OV-17 on Chromosorb W AW DMCS at 130 degrees C. Determination was possible with 3-100 micrograms of the dehydrated reaction mixture. The procedure for determining nicotinamide in various meats and meat products involves direct analysis by GLC-FID without a clean-up stage; the detection limit is 5 ppm and the recovery ranged from 93.4 to 104.6% (average 98.0%). Various possible interferents in the samples did not interfere in the production or determination of 3-cyanopyridine. The procedure is suitable for routine use. The dehydrated derivative of nicotinamide was confirmed as 3-cyanopyridine by combined gas chromatography-mass spectrometry and infrared spectrometry.

Simultaneous liquid chromatographic determination of residual synthetic antibacterials in cultured fish.

A liquid chromatographic method is described to determine simultaneously the following 11 synthetic antibacterial agents used in a fishery: nitrofuran derivatives furazolidone, nifurpirinol, difurazone, and furamizole; sulfa drugs sulfamerazine, sulfisozole, sulfamonomethoxine, and sulfadimethoxine; and, oxolinic, nalidixic, and piromidic acids. A Nucleosil C18 column was used with tetrahydrofuran-acetonitrile-phosphoric acid-water (29 + 1 + 0.06 + 69.94) as the mobile phase. Pretreatment of the fish meat sample with acetone extraction and alumina column cleanup gave good separation of the LC peaks without interference from any other components. Recovery of the antibacterial agents was ca 80%. The lower limit of detection of the drugs was 1-2 ng for 10 microL injection.

Simultaneous determination of nalidixic acid, oxolinic acid and piromidic acid in fish by high-performance liquid chromatography with fluorescence and UV detection.

A simple and rapid method for the simultaneous determination of nalidixic acid (NA), oxolinic acid (OXA) and piromidic acid (PMA) in cultured fish has been developed by high-performance liquid chromatography (HPLC). The drugs were extracted with 0.1% metaphosphoric acid-methanol (6:4), followed by a Sep-Pak C18 clean-up procedure. The HPLC separation was carried out on a Kaseisorb LC ODS 300-5 column (25 cm x 4.6 mm I.D.) using 5 mM phosphate buffer-acetonitrile (6:4) as a mobile phase. A fluorescence detector was used for NA and OXA at the excitation wavelength of 325 nm and the emission wavelength of 365 nm and an ultraviolet detector at 280 nm for PMA. The calibration graphs were rectilinear from 1 to 10 ng for OXA, from 2 to 20 ng for NA and PMA. The recoveries of NA, OXA and PMA added to fish were 81.5-85.3, 83.7-88.7 and 80.9-84.9%, respectively, with high accuracy. The limits of detection were 0.01 micrograms/g for each drug.

Determination of the stable isotope of nitrite flux in the blood of mice by gas chromatography-mass spectrometry with selected ion monitoring.

A simple and reliable method for the determination of the stable isotope of nitrite (15NO2) flux in the blood of mice is described. It is based on the reaction of 15NO2 with 1-hydrazinophthalazine in acidic solution to form [15N]tetrazolophthalazine, a stable compound which can be extracted with an organic solvent and then determined by gas chromatography-mass spectrometry with selected ion monitoring using the pentafluorobenzoyl ester of 2,4-dinitro-6-sec.-butylphenol as an internal standard. Amounts of 0.2-10 micrograms of 15NO2 can be determined. The detection limit of 15NO2 was 0.1 microgram/ml. This is a specific method for 15NO2. The procedure for determining 15NO2 in the blood of mice involves extraction with solvent, followed by further clean-up by alumina column chromatography; the detection limit is 0.05 microgram. With the new technique we were able to perform a metabolic fate study of nitrite in the blood of mice following a single oral dose of 15NO2 or the stable isotope of nitrate (15NO3).

Gas-liquid chromatographic determination of trace amounts of nitrite in egg, egg white, and egg yolk.

A simple, sensitive, and practical method is described for determination of nitrite in egg, egg white, and egg yolk. Egg is deproteinized by adding a mixture of ammonium thiocyanate, mercuric chloride, and zinc acetate, and centrifuged. Nitrite in the supernate is converted to tetrazolophthalazine by reaction with hydralazine in acidic solution and then determined by gas-liquid chromatography with an electron-capture detector (GLC-ECD) and a column of OV-225 on Chromosorb W(HP). Nitrite concentrations from 5 to 50 ng/mL are calculated from peak height; the detection limit is 3 ng/mL extract. Recoveries from eggs, egg whites, and egg yolks ranged from 91.7 to 98.0%. The mean nitrite concentration in 50 egg samples was 0.04 ppm (0.01-0.11 ppm) with a detection limit of 4 ng nitrite/g.