Timing of insemination and fertility in dairy and beef cattle receiving timed artificial insemination using sex-sorted sperm.

The objective was to evaluate the effects of timing of insemination and type of semen in cattle subjected to timed artificial insemination (TAI). In Experiment 1, 420 cyclic Jersey heifers were bred at either 54 or 60 h after P4-device removal, using either sex-sorted (2.1 × 10(6) sperm/straw) or non-sorted sperm (20 × 10(6) sperm/straw) from three sires (2 × 2 factorial design). There was an interaction (P = 0.06) between time of AI and type of semen on pregnancy per AI (P/AI, at 30 to 42 d after TAI); it was greater when sex-sorted sperm (P < 0.01) was used at 60 h (31.4%; 32/102) than at 54 h (16.2%; 17/105). In contrast, altering the timing of AI did not affect conception results with non-sorted sperm (54 h = 50.5%; 51/101 versus 60 h = 51.8%; 58/112; P = 0.95). There was an effect of sire (P < 0.01) on P/AI, but no interaction between sire and time of AI (P = 0.88). In Experiment 2, 389 suckled Bos indicus beef cows were enrolled in the same treatment groups used in Experiment 1. Sex-sorted sperm resulted in lower P/AI (41.8%; 82/196; P = 0.05) than non-sorted sperm (51.8%; 100/193). In addition, there was a tendency for greater P/AI (P = 0.11) when TAI was performed 60 h (50.8%; 99/195) versus 54 h (42.8%; 83/194) after removing the progestin implant. In Experiment 3, 339 suckled B. indicus cows were randomly assigned to receive TAI with sex-sorted sperm at 36, 48, or 60 h after P4 device removal. Ultrasonographic examinations were performed twice daily in all cows to confirm ovulation. On average, ovulation occurred 71.8 ± 7.8 h after P4 removal, and greater P/AI was achieved when insemination was performed closer to ovulation. The P/AI was greatest (37.9%) for TAI performed between 0 and 12 h before ovulation, whereas P/AI was significantly less for TAI performed between 12.1 and 24 h (19.4%) or >24 h (5.8%) before ovulation. In conclusion, sex-sorted sperm resulted in a lesser P/AI than non-sorted sperm following TAI. However, improvements in P/AI with delayed time of AI were possible (Experiments 1 and 3), and seemed achievable when breeding at 60 h following progestin implant removal, compared to the standard 54 h normally used in TAI protocols.

Special care dentistry: Midazolam conscious sedation for patients with neurological diseases.

AIM: Midazolam is used very often to control the anxiety of patients for dental treatment, especially in patients with special needs. The objective of this study was to evaluate the efficiency of Midazolam in patients with neurological diseases referred for dental treatment. STUDY DESIGN: Descriptive study. MATERIALS AND METHODS: Forty consecutive patients with neurological disorders (encephalopathy, autism, and epilepsy) were referred to dental treatment, and 45 sedations were performed; all were sedated with Midazolam (intramuscular 0.2-0.3 mg/kg or intravenous 0.1mg/kg) and all were anesthetised with lidocaine 2% (0.5-2 mL). During the dental procedure, their behavior was analysed and classified into 3 categories: A (indifferent), B (reacted but allowed treatment), and C (did not allow treatment). Data were tabbed and statistically analysed. RESULTS: The final patients' classification was: A 22 (49%), B 18 (40%) and C 5 (11%); the patients with encephalopathy had the best results of sedation according to the proposed classification (p<0.05). CONCLUSION: Midazolam demonstrated to be effective in 89% of this sample for dental procedures in patients with neurological and behavioral disturbances, but it was less effective for patients with autism (p<0.05).

Morphological alterations in the kidney of rats with natural and experimental Leptospira infection.

Leptospirosis is a widespread anthropozoonosis, with a broad array of mammalian reservoirs, occurring as rural endemics, urban outbreaks related to floods, and emergent disease associated with water sports and recreational exposure in developed countries. Rats are the major source of human infection, particularly in urban areas; however few reports have focused on the pathology of leptospirosis in this host. This study reports pathological changes in 60 kidneys from captured wild rats and compares these with changes in the kidney of Wistar rats experimentally infected with Leptospira interrogans serovar Copenhageni strain FIOCRUZ L1-130. A broad range of morphological alterations were detected in the kidneys from captured rats but interstitial nephritis was the only feature reproduced under experimental conditions. The role of interstitial nephritis in the pathogenesis of leptospirosis is reviewed and it is suggested that rats may provide a potential tool for the study of colonization mechanisms and host resistance in acute leptospiral disease.

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50 percent of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1 percent Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies

Immunocytochemical and biochemical detection of alpha-L-fucosidase in Trypanosoma cruzi.

The aim of the present study was to demonstrate the presence of alpha-L-fucosidase in Trypanosoma cruzi. Immunocytochemical and biochemical techniques were used to localize and characterize a membrane-associated, neutral-pH-optimum, alpha-L-fucosidase from the parasite. Light and electron microscopy localized the alpha-L-fucosidase specifically on the surface of the parasite and on membranes in the posterior region of the epimastigote stage. Although much less intense, labeling was also detected on the surface of trypomastigotes. At least 50% of the alpha-L-fucosidase activity was associated with epimastigote membrane solubilized with 1 M NaCl or 1% Triton X-100, suggesting that alpha-L-fucosidase is peripherally associated with membranes. The enzyme from epimastigotes had a neutral pH optimum (near 7) but displayed low specific activity when p-nitrophenyl-alpha-L-fucoside was employed as substrate (0.028 U/mg protein for epimastigotes and 0.015 U/mg protein for tissue culture-derived trypomastigotes). Polyacrylamide gel electrophoresis and Western blotting analysis both showed an expected 50-kDa polypeptide which was immunoreactive with anti-alpha-L-fucosidase antibodies.

Trypanosoma cruzi: characterization of an intracellular epimastigote-like form.

Almeida-de-Faria, M., Freymüller, E., Colli, W., and Alves, M. J. M. 1999. Trypanosoma cruzi: Characterization of an intracellular epimastigote-like form. Experimental Parasitology 92, 263-274. A detailed study of transient epimastigote-like forms as intermediates in the differentiation of Trypanosoma cruzi amastigotes to trypomastigotes inside the host cell cytoplasm was undertaken using the CL-14 clone grown in cells maintained at 33 degrees C. Several parameters related to these forms have been compared with epimastigotes and other stages of the parasite. Consequently, the designation of intracellular epimastigotes is proposed for these forms. Despite being five times shorter (5.4 +/- 0.7 micrometer) than the extracellular epimastigote (25.2 +/- 2.1 micrometer), the overall morphology of the intracellular epimastigote is very similar to a bona fide epimastigote, when cell shape, position, and general aspect of organelles are compared by transmission electron microscopy. Epimastigotes from both sources are lysed by human complement and bind to DEAE-cellulose, in contrast to amastigotes and trypomastigote forms. A monoclonal antibody (3C5) reacts with both epimastigotes either isolated from axenic media or intracellular and very faintly with amastigotes, but not with trypomastigotes. Some differences of a quantitative nature are apparent between the two epimastigote forms when reactivities with lectins or stage-specific antibodies are compared, revealing the transient nature of the intracellular epimastigote. The epitope recognized by 3C5 monoclonal antibody reacts slightly more intensely with extracellular than with intracellular epimastigotes, as detected by immunoelectron microscopy. Also a very faint reaction of the intracellular epimastigotes was observed with monoclonal antibody 2C2, an antibody which recognizes a glycoprotein specific for the amastigote stage. Biological parameters as growth curves in axenic media and inhability to invade nonphagocytic tissue-cultured cells are similar in the epimastigotes from both origins. It is proposed that the epimastigote-like forms are an obligatory transitional stage in the transformation of amastigotes to trypomastigotes with a variable time of permanency in the host cell cytoplasm depending on environmental conditions.

Development of NADPH-diaphorase in the avian retina: regulation by calcium ions and relation to nitric oxide synthase.

Nitric oxide plays an important role as an intercellular messenger in the CNS. In the present work we measured NADPH-diaphorase activity, which is considered to be a marker of cells producing nitric oxide, in homogenates of the developing chick retina. The enzyme activity can be detected beginning in 8-day-old embryonic retinas with no further quantitative variations throughout development. Arginine analogues inhibit approximately 65% of the activity in embryonic retinas and 50% in posthatched retinas. The enzyme is stimulated 50% by 2 mM calcium chloride in retinas from 8 to 14 embryonic days, but this effect decreases to 20% in 17-day embryonic retinas and practically disappears in posthatched animals. The stimulation by calcium is completely blocked by arginine analogues. The decrease in enzyme activity at posthatched retinas is not due to stimulation by endogenous calcium or the presence of insufficient amounts of calmodulin, because addition of EGTA or calmodulin, respectively, did not restore the stimulation to levels observed at embryonic stages. Inhibition of NADPH-diaphorase activity by NG-nitro-L-arginine or L-NG-(iminoethyl) ornithine is concentration dependent with IC50 values of approximately 1 mM at all stages studied. However, in the presence of calcium, the inhibition by both analogues is shifted to the left and is apparently biphasic at all developmental stages, including in posthatched animals, with IC50 values in the low micromolar range. NADPH-diaphorase was also detected by histochemistry in specific groups of cells in the early embryonic retina and in subsets of amacrine and ganglion cells, as well as in photoreceptors, in more developed retinas. The results indicate that different isoforms of nitric oxide synthase are present in the chick retina and that a calcium-dependent isoform is predominant in early periods of development.

Biochemical characterization of NADPH-diaphorase in the chick embryo retina: stimulation by calcium ions and inhibition by arginine analogs.

Nitric oxide is an important intercellular messenger in the central nervous system. NADPH-diaphorase, reported to be identical to nitric oxide synthase, is present in specific groups of cells in several neural tissues, including the retina. We determined NADPH-diaphorase activity in homogenates of the chick embryo retina. The enzyme activity was measured spectrophotometrically at 585 nm after incubating retinal total homogenates (100-150 micrograms protein) with 1 mM NADPH and 0.5 mM nitroblue tetrazolium in 50 mM Tris buffer, pH 8.1, at 37 degrees C. NADPH-diaphorase was detected in 14-day old retinas and 53-65% of the enzyme activity was inhibited by 3 mM NG-nitro-L-arginine (NARG), the arginine analog. One mM L-N5-(1-iminoethyl)ornithine (NIO) was the most potent inhibitor (63% inhibition) while 3 mM NG-nitro-L-arginine methyl ester (NAME) (33% inhibition) and 1 mM NG-monomethyl-L-arginine acetate (NMMA) (14% inhibition) were less effective. Enzyme activity was increased by 48% by 2 mM calcium chloride, an effect reversed by 1 mM EGTA or EDTA. Basal enzyme levels were also partially inhibited by the chelators, indicating the presence of calcium-dependent and -independent isoforms of nitric oxide synthase in the retina. The results show that the NADPH-diaphorase assay is simple and sensitive and that the different isoforms of nitric oxide synthase expressed in chick retinal cells during development can be demonstrated.

[Comparison of negative chronotropic action of nitrendipine, nifedipine and verapamil on the isolated right atrium of normotensive and renovascular hypertensive rats]. / Comparação do cronotropismo negativo da nitrendipina, nifedipina e verapamil no átrio direito isolado de ratos normotensos e com hipertensão renovascular.

PURPOSE: To study the negative chronotropic effects of nitrendipine, nifedipine and verapamil in isolated right atria from normotensive and renovascular hypertensive rats. MATERIAL AND METHODS: Hypertension (medium arterial pressure, MAP = 154 +/- 4 mmHg) was induced by applying a silver clip to the left renal artery and right nephrectomy. Control rats (MAP = 109 +/- 2 mmHg) were submitted to right nephrectomy only. The animals were studied 2 weeks after surgery. Different preparations were used to obtain cumulative dose-response curves (0.01 microM to 100 microM) with each drug. RESULTS: No difference in "in vitro" initial sinusal rate of control (243 +/- 7 bpm) and hypertensive (245 +/- 5 bpm) rats was observed. The negative chronotropic response produced by calcium channel blockers was similar in normotensive and hypertensive groups. 2 microM verapamil, 4 microM nitrendipine or 20 microM nifedipine was necessary to produce a 50% decrease of the initial sinusal rate. CONCLUSION: Sensitivity of the sinusal pacemaker to calcium antagonists does not change in this model of hypertension. Moreover, the negative chronotropic effect of nitrendipine is stronger than nifedipine and weaker than verapamil.

Swimming escape learning in rats with telencephalic lesions.

Experiments were conducted to determine whether rats having unilateral or bilateral ablation of either the neocortex or telencephalon can learn to escape from a tank of water by climbing a platform. Hemi- or bilaterally decorticated rats were able to learn the swimming escape task, the same being observed for hemidetelencephalated rats. Fully detelencephalated rats, however, did not exhibit this capability. These results show that integrity of one of the cerebral hemispheres is necessary in order to allow swimming escape learning.