A method was developed for percutaneous ultrasound-guided cholecystocentesis in cattle. The procedure was performed on the right side in the 9th, 10th, or 11th intercostal space of 30 cows. Of the 30 cows, 20 were slaughtered 24 hours after cholecystocentesis and the remaining 10 cows were slaughtered after a 10-day observation period. Changes in the peritoneum and gallbladder wall, observed at slaughter, were minimal. During the 10-day observation period, general behavior, attitude, and appetite of the 10 cows were normal. A transient, slight increase in rectal temperature was observed in 6 cows at 4, 5, or 8 days after cholecystocentesis. Total and differential WBC counts and total protein and fibrinogen concentrations, determined daily, were all within normal ranges. Bile samples from 20 cows were examined microscopically and biochemically. Fasciola hepatica and Dicrocoelium dendriticum eggs were observed in bile from 7 and 12 cows, respectively. Fecal examination revealed F hepatica eggs in 4 cows; D dendriticum eggs were not identified in any of the fecal samples. In 1 cow, F hepatica eggs were observed in the feces, but not in the bile. Bile acids concentration in bile varied from 12.5 to 68.5 mmol/L (mean +/- SD, 45.3 +/- 3.05 mmol/l) and in serum from 3.8 to 281.0 micromol/l (41.6 +/- 17.24 micromol/L). Negative correlation was obtained between bile acids concentration in bile and that in serum (r = - 0.60, P < 0.01). It was concluded that percutaneous ultrasound-guided cholecystocentesis in cows is a safe procedure and that microscopic and biochemical examinations of obtained bile can be useful diagnostic aids.

This study was carried out to develop a newly designed ovum pick-up(OPU) instrument for ultrasound-guided transvaginal follicular aspiration in cows. This new instrument consists of out- & inner-layer stainless pipes and a grip with a trigger(hand ) switch. Some gauge types of disposable needles and tubes can be attached to this inner pipe. With this instrument, while grasping an avary with one hand the other hand can handle in apiration and vacuum on/off with the least assitant's help. with this instrument the mean recovery rate of bovine follicular oocytes was 45.2%. In recovered oocytes, usable oocytes(Grade I & II) were 30.4% and this rate meant 1.4 oocytes per ovary. For 30 days after initial aspiration with this instrument, some adverse effects such as adhesion, hemorrhage, hematoma and other mass formation in/with ovaries were also examined by rectal examination, ultrasonographic and endoscopic images. Adhesion was found in one ovary 1 week after aspiration, and hemorrhagic lesion was found 1-2 days and petechia were found 3-5 days after aspiration and there was no remarkable adverse effects. It was found that this instrument could be applicable and safe for ovum pick-up in cows.

To establish whether Mycobacterium paratuberculosis could be cultured from Dulbecco phosphate-buffered saline solution (DPBSS) and to test 3 sampling methods, DPBSS supplemented with 2% fetal bovine serum was inoculated with M paratuberculosis at concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units/ml. The inoculated media was sampled after mixing, after centrifugation, and after centrifugation and decontamination with 0.75% hexadecylpyridinium chloride. The samples were inoculated onto 3 slants of Herrolds egg yolk medium supplemented with sodium pyruvate and mycobactin J and 1 slant without mycobactin J. Mycobacterium paratuberculosis was isolated following all 3 sampling methods for all concentrations. Treatment with hexadecylpyridinium chloride decreased the number of colonies isolated. To test the efficacy of a 10-step wash procedure for removing M paratuberculosis from bovine ova, washed zona pellucida intact bovine ova were incubated in DPBSS supplemented with 2% fetal bovine serum containing concentrations of 10(4), 10(3), 10(2), 10(1), and 10(0) colony-forming units of M paratuberculosis/ml for 12 hours at 22 C. Ten zona pellucida intact ova were removed from each concentration and washed by passing through 10 changes of DPBSS supplemented with 15% fetal bovine serum. The media from each wash step was inoculated onto slants of Herrolds egg yolk medium. The ova were included with the tenth wash step. Mycobacterium paratuberculosis was isolated from 1 of 10 tenth-wash steps at the 10(4) concentration and 5 of 10 tenth-wash steps at 10(3).

Effects of oxygen (O2) tension in the gas atmosphere during in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) on the efficiency of in vitro production of mouse embryos were examined. Mouse oocytes recovered from large antral follicles were subjected to IVM in Waymouth medium for 15, 16 and 17 hr under 5 or 20% O2 and then subjected to IVF and IVC under 5 or 20% O2 tension. Lowering the O2 tension in the gas atmosphere for IVM from 20 to 5% improved the cleavage rate after IVF when the oocytes were subjected to IVM for 15 hr; however, no improvement in the cleavage rate was observed when the culture period for IVM was extended to 16 and 17 hr. Lowering the O2 tension to 5% for IVM and IVC improved the development of the cleaved oocytes to the blastocyst stage, regardless of the culture period for IVM. However, the O2 tension for IVF had no remarkable effect on the subsequent embryonic development. These results demonstrate that 5% O2 is superior to 20% O2 for IVM and IVC, and suggest that 20% O2 for IVM may delay oocyte maturation and/or the acquisition of fertilizability and impair the developmental competence of oocytes.

Mouse metaphase two (M two) oocytes were exposed to 50 mug/ml etoposide (ETO) before and after parthenogenetic activation with 7% ethanol and they were washed with 0.75 M sucrose. The ETO treated parthenogenetically activated oocytes were cultured or fused to single blastomeres of late 2-cell stage mouse embryo to test their ability to support development in vitro. In parallel untreated parthenogenetically activated oocytes were cultured to serve as control. None of ETO treated oocytes developed beyond the 2-cell stage, whereas 4% of the reconstituted embryos and 35% of control developed to blastocysts. It is concluded that mouse M two oocytes can be functionally enucleated by ETO treatment and can be used for nuclear transfer experiments