Chlorpyrifos and endosulfan affect buffalo oocyte maturation, fertilization, and embryo development in vitro directly and through cumulus cells.

This study was undertaken to examine the effect of 10 different levels (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 2.0, and 4.0 µg/mL) of two pesticides (chlorpyrifos and endosulfan) on buffalo oocyte viability, maturation, fertilization, and developmental competences in vitro. Studies were conducted to test the development of oocytes cultured with pesticides during maturation, fertilization, and during different embryo development stages. We also conducted experiments to test the hypotheses that the effects of these pesticides are hormones and somatic cells mediated. We observed a dose dependent decline in viability and developmental competence rates of oocytes. Chlorpyrifos and endosulfan had a negative impact on oocytes at 0.02 and 0.1 µg/mL levels, respectively. These pesticides reduced the oocyte nuclear maturation by a direct effect on oocytes, cumulus cell-mediated action, and by blocking the action of hormones. Chlorpyrifos was found to be more ovotoxic and embryotoxic than endosulfan. This study will provide information on dose-response relationship and risk assessment in domestic buffaloes.

Effects of exposure to heavy metals on viability, maturation, fertilization, and embryonic development of buffalo (Bubalus bubalis) oocytes in vitro.

The aim of the present study was to examine the effect of heavy metals, cadmium and lead, on buffalo oocyte viability and in vitro development. Oocytes were aspirated from ovaries of slaughtered buffaloes. Only viable and metabolically active oocytes with more than three layers of cumulus cell layers and homogeneous ooplasm were selected. Effects of nine concentrations (0, 0.005, 0.05, 0.5, 1.0, 1.5, 2.5, 5, and 10 microg/mL) of cadmium or lead on buffalo oocyte viability, morphological abnormities, maturation, and embryonic development in vitro were studied. Oocytes were cultured for 24 h and then checked for viability (0.05% trypan blue staining for 2 min), morphological abnormalities, and reduction assay by MTT test in experiment 1. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were determined (experiment 2). In experiment 3, viable oocytes were matured in vitro in media containing different levels of cadmium or lead and then inseminated in vitro with frozen-thawed spermatozoa, and the resultant cleaved embryos were cultured in a control embryo culture medium for 8 days. In experiment 4, oocytes were cultured in control oocyte maturation medium, then fertilized, and the resultant embryos were cultured in media containing different levels of cadmium or lead for 8 days. The number of cells in the trophectoderm and inner cell mass (ICM) and the total cell counts (TCN) of blastocysts derived by in vitro culture of two- to four-cell-stage embryos (produced in control medium) in media containing 0, 0.005, 0.05, 0.5, and 1.0 microg/mL of cadmium or lead were analyzed by differential staining technique (experiment 5). Cadmium and lead were found to have a dose-dependent effect on viability, morphological abnormities, maturation, cleavage and morula/blastocyst yield, and blastocyst hatching. A significant decline in viability of oocytes was observed at 1.0 mg/mL cadmium or lead compared to the control group. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were 18 and 32 microg/mL, respectively. Cadmium and lead at 1.0 and 2.5 microg/mL, respectively, caused a significant reduction of maturation of oocytes compared to the lower concentrations. No cleavage or morulae/blastocysts were produced when the oocytes/embryos were cultured in media containing 2.5 and 5.0 mg/mL of either cadmium or lead, respectively. Similarly, no morulae/blastocysts were produced from cleaved embryos cultured in media containing 2.5 and 5.0 microg/mL cadmium and lead, respectively. The developmental block, degeneration, and asynchronous divisions were higher in embryos exposed to cadmium than in those exposed to lead. TCN and number of cells in ICM were significantly lower in blastocysts derived from two- to four-cell-stage embryos cultured in media containing heavy metals. In conclusion, cadmium and lead lowered the viability and development of buffalo oocytes but at a concentration higher than that estimated in the body fluids and environment. Cadmium was found to be more ovotoxic than lead.

Tyrosine phosphorylation of a 38-kDa capacitation-associated buffalo (Bubalus bubalis) sperm protein is induced by L-arginine and regulated through a cAMP/PKA-independent pathway.

The aim of the present study was to determine the effect of L-arginine on nitric oxide (NO*) synthesis, capacitation and protein tyrosine phosphorylation in buffalo spermatozoa. Ejaculated buffalo spermatozoa were capacitated in the absence or presence of heparin, or L-arginine or N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase (NOS) for 6 h. Capacitating spermatozoa generated NO* both spontaneously and following stimulation with L-arginine and L-NAME quenched such L-arginine-induced NO* production. Immunolocalization of NOS suggested for existence of constitutive NOS in buffalo spermatozoa. L-Arginine (10 mm) was found to be a potent capacitating agent and addition of L-NAME to the incubation media attenuated both L-arginine and heparin-induced capacitation and suggested that NO* is involved in the capacitation of buffalo spermatozoa. Two sperm proteins of M(r) 38 000 (p38) and 20 000 (p20) were tyrosine phosphorylated extensively by both heparin and L-arginine. Of these, the tyrosine phosphorylation of p38 was insensitive to both induction by cAMP agonists as well as inhibition by a protein kinase A (PKA) inhibitor. Further, most of these L-arginine-induced tyrosine phosphorylated proteins were localized to the midpiece and principal piece regions of flagellum of capacitated spermatozoa and suggested that sperm flagellum takes active part during capacitation. These results indicated that L-arginine induces capacitation of buffalo spermatozoa through NO* synthesis and tyrosine phosphorylation of specific sperm proteins involving a pathway independent of cAMP/PKA.

Effect of reactive oxygen species on capacitation and associated protein tyrosine phosphorylation in buffalo (Bubalus bubalis) spermatozoa.

In the present study, the effect of two particular reactive oxygen species (ROS), superoxide anion (O(2)(-)) and hydrogen peroxide (H(2)O(2)) on buffalo (Bubalus bubalis) sperm capacitation and associated protein tyrosine phosphorylation was studied. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50 x 10(6)/mL and incubated at 38.5 degrees C for 6h with or without heparin (10(g/mL; a positive control), or xanthine (X; 0.5mM)-xanthine oxidase (XO; 0.05 U/mL)-catalase (C; 2100 U/mL) system that generates O(2)(-) or NADPH (5mM) that stimulates the endogenous O(2)(-) production or H(2)O(2) (50 microM). The specific effect of O(2)(-), H(2)O(2) and NADPH on buffalo sperm capacitation and protein tyrosine phosphorylation was assessed by the addition of superoxide dismutase (SOD), catalase and diphenylene iodonium (DPI), respectively, to the incubation medium. Each of X+XO+C system, NADPH and H(2)O(2) induced a significantly higher percentage (P<0.05) of capacitation in buffalo spermatozoa compared to control. However, DPI inhibited this NADPH-induced capacitation and protein tyrosine phosphorylation and suggested for existence of an oxidase in buffalo spermatozoa. Using immunoblotting technique, at least seven tyrosine-phosphorylated proteins (20, 32, 38, 45, 49, 78 and 95 kDa) were detected in capacitated buffalo spermatozoa. Out of these, the tyrosine phosphorylation of p95 was induced extensively by both O(2)(-) as well as exogenous source of H(2)O(2) and using specific activators and inhibitors of signaling pathways, it was found this induction was regulated through a cAMP-dependent PKA pathway. Further, immunofluorescent localization study revealed that these ROS-induced tyrosine-phosphorylated proteins are mostly distributed in the midpiece and principal piece regions of the flagellum of capacitated spermatozoa and suggested for increased molecular activity in flagellum during capacitation. Thus, the study revealed that both O(2)(-) and H(2)O(2) promote capacitation and associated protein tyrosine phosphorylation in buffalo spermatozoa and unlike human and bovine, a different subset of sperm proteins were tyrosine-phosphorylated during heparin- and ROS-induced capacitation and regulation of these ROS-induced processes were mediated through a cAMP/PKA signaling pathway.

Production of superoxide anion and hydrogen peroxide by capacitating buffalo (Bubalus bubalis) spermatozoa.

In the present study attempts were made to detect and quantify the generation of superoxide anion (O(2)(*-)) and hydrogen peroxide (H(2)O(2)) by capacitating buffalo spermatozoa. Ejaculated buffalo spermatozoa were suspended in sp-TALP medium at 50x10(6)mL(-1) and incubated at 38.5 degrees C with 5% CO(2) in air in the absence or presence of heparin (a capacitation inducer) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) or diphenyleneiodonium (DPI, a flavoprotein inhibitor) for 6h. Production rate of O(2)(*-) and H(2)O(2) by spermatozoa at different hours of capacitation were measured by cytochrome c reduction and phenol red oxidation assays, respectively. Spermatozoa generated both O(2)(*-) and H(2)O(2) spontaneously and following stimulation with heparin and a significant increase of O(2)(*-) production was observed in the presence of NADPH. However, DPI inhibited this NADPH-induced O(2)(*-) production and suggested for existence of putative NADPH-oxidase that constitute a specific O(2)(*-) generating systems in buffalo spermatozoa. Results of our study indicated that buffalo spermatozoa generate O(2)(*-) and H(2)O(2) and production of these free radicals is induced during capacitation.

Shielding for neutron scattered dose to the fetus in patients treated with 18 MV x-ray beams.

Neutrons are associated with therapeutic high energy x-ray beams as a contaminant that contributes significant unwanted dose to the patient. Measurement of both photon and neutron scattered dose at the position of a fetus from chest irradiation by a large field 18 MV x-ray beam was performed using an ionization chamber and superheated drop detector, respectively. Shielding construction to reduce this scattered dose was investigated using both lead sheet and borated polyethylene slabs. A 7.35 cm lead shield reduced the scattered photon dose by 50% and the scattered neutron dose by 40%. Adding 10 cm of 5% borated polyethylene to this lead shield reduced the scattered neutron dose by a factor of 7.5 from the unshielded value. When the 5% borated polyethylene was replaced by the same thickness of 30% borated polyethylene there was no significant change in the reduction of neutron scatter dose. The most efficient shield studied reduced the neutron scatter dose by a factor of 10. The results indicate that most of the scattered neutrons present at the position of the fetus produced by an 18 MV x-ray beam are of low energy and in the thermal to 0.57 MeV range since lead is almost transparent to neutrons with energies lower than 0.57 MeV. This article constitutes the first report of an effective shield to reduce neutron dose at the fetus when treating a pregnant woman with a high energy x-ray beam.

Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves.

Outer membrane protein (OMP) from Pasteurella multocida serotype B:2 was extracted and characterized using SDS-PAGE. Ten major polypeptide bands of MW 88 to 25 kDa were observed. Immunoblotting suggested that the polypeptides of MW 44, 37 and 30 kDa were the major immunogens. Buffalo calves vaccinated with the OMP vaccine or a commercial haemorrhagic septicaemia oil adjuvant vaccine developed highest mean log10 ELISA titres day 21 postvaccination (pv). Antibody titres detectable in these animals using an indirect haemagglutination assay were lower than the ELISA titres but the pattern of the antibody response was similar. A passive mouse protection assay revealed that the maximum protection against the challenge infection was conferred by the serum collected from both the groups on day 21 pv and 26 pv. Following challenge with virulent P. multocida cells, all the five OMP vaccinated animals survived whereas only 2 out of the 3 HS oil adjuvant vaccinated animals withstood the challenge. Results suggested that OMP was protective and could be used in vaccines against haemorrhagic septicaemia.

Effect of Pasteurella multocida vaccination on buffalo polymorphonuclear hydrogen peroxide and nitric oxide production.

An attempt was made to investigate the effect of Pasteurella multocida on certain microbicidal reactive oxygen and nitrogen intermediates released by the polymorphonuclear cells (PMNs) from the vaccinated animals. The PMNs from the peripheral blood of both control and experimental buffaloes vaccinated against haemorrhagic septicaemia were isolated. PMNs from control animals upon activation with P. multocida lipopolysaccharide (LPS) and live P. multocida cells generated higher levels of hydrogen peroxide (H2O2) and nitric oxide (NO-) than the non-activated cells (P < 0.01). In the presence of P. multocida LPS, PMNs from animals vaccinated against haemorrhagic septicaemia generated significantly higher H2O2 (P < 0.05) and NO- (P < 0.01) than the PMNs from control animals. L-Arginine when added to the activation medium enhanced the production of NO- in a dose-dependent manner. This indicated the role of arginine in NO- production. The study suggested that buffalo PMNs possessed a potent oxidant defence system even in the presence of P. multocida, an antiphagocytic bacterium.

The content, integrity, and efficacy of a nurse coaching intervention in type 2 diabetes.

PURPOSE: The purpose of this study was to systematically evaluate the content, integrity, and efficacy of a nurse coaching intervention provided after diabetes education that focused on dietary and exercise lifestyle change in persons with type 2 diabetes. METHODS: A multimethod design incorporated an interpretive approach to examine the content and integrity of the intervention and a multiple-baseline, single-subject method to determine the preliminary efficacy of the intervention. RESULTS: The primary strategies of the nurse coaching intervention consisted of facilitating lifestyle change through educational reinforcement, psychosocial support, and motivational guidance. Aggregate quantitative outcomes revealed a modest increase in health-promoting behaviors and a decrease in fasting blood glucose, indicating a trend toward physiologic adaptation. Participants demonstrated a significant increase in integration reflective of psychosocial adaptation. CONCLUSIONS: Providing individualized nursing care after diabetes education may improve health outcomes and the quality of life of persons newly diagnosed with type 2 diabetes. This multimethod design is a cost-effective approach for preliminary evaluation of complex and/or novel interventions.

An inducible riboflavin synthetase from a pseudomonad.

Riboflavin synthetase activity is induced in a strain of Pseudomonas fluorescens-putida intermediate only under conditions permitting an accumulation of compound P, a 2,4-dioxopteridine, in the medium. The effect of different amino acids and sugars on the production of compound P and riboflavin synthetase was determined. The enzyme was partially destroyed by ammonium sulphate fractionation. It is inhibited by heavy metal ions and PCMB. PCMB inhibition can be almost completely reversed by GSH.

A sensitive neutron dosimeter using superheated liquid.

The present work relates to a sensitive neutron dosimeter, a device for monitoring neutron dose in some accelerator and reactor sites. This device is capable of measuring a neutron dose as small as 0.1 microSv using superheated liquid as a sensitive liquid. The nucleation was measured by the volumetric method developed in our laboratory. The dose response of superheated drops of four liquids having boiling points of 8.92, -29.79, -40.75 and -45.6 degrees C, irradiated by a 3 Ci Am-Be neutron source has also been presented in this article.

Electrophoretic detection of myeloperoxidase, protease, lactoferrin and lysozyme in buffalo polymorphonuclear granular acid extracts.

Polymorphonuclear (PMN) cells of more than 90% viability and 92% purity were isolated from the peripheral blood of buffaloes. The cationic proteins were extracted with 0.2 mol/L sodium acetate, pH 4.0 from the granules in the PMN and subjected to both non-denaturing and denaturing acid urea polyacrylamide gel electrophoresis (AUPAGE) for identification of myeloperoxidase (MPO), lysozyme, protease activity and lactoferrin. Protease was identified using alpha-naphthyl acetate as substrate, while lactoferrin was identified using a reference lactoferrin from bovine milk in AUPAGE, and by double immunodiffusion and Western blot techniques. Based on AUPAGE, lysozyme was found to be most cationic of all the proteins and peptides from the PMN granules as was evident from reference lysozyme run. The results indicated that the granules in buffalo PMN cells have lysozyme, protease, MPO and lactoferrin.

Extent of utilisation of maternal care services of P.H.C. by the families of a rural area.

PIP: The utilization of the maternal health care services offered by an upgraded primary health care (PHC) facility in a rural area of West Bengal, India was assessed. Information on the use of the maternal services by pregnant women over a 5-year period was collected from a house-to-house sample of 100 families living less than 1 hour away from the health facility and having at least 1 child born into the family in the previous 5-year period. Women in 58% of the families used the prenatal services of the facility, 6% received prenatal care from private practitioners, and 36% received no prenatal care. Reasons given for not using the facility were 1) using the clinic was too time consuming, 2) the staff was unfriendly, 3) a lack of interest in the services provided. There was no significant differences between prenatal service utililizers and nonuser in regard to caste differences. Utilizers were somewhat more likely to live in households with a literate household head than nonusers. The number of visits made by the utilizers ranged from 1-5, but many respondents had difficulty recalling the exact number. Utilizers were no more likely than nonusers to use the delivery services of the PHC. Among the 58 women who used either the prenatal services of the PHC or of private practitioners, 34 had their deliveries at the PHC, 23 at home and 1 in the hospital. Among the 42 women who received no prenatal care, 15 gave birth at the PHC center, 20 at home, and 4 at nursing homes. Home deliveries were conducted either by untrained midwives or by family members. 3 cases of neonatal tetanus and 1 case of maternal tetanus were reported in the community during the 5 year period. All of these births occurred at home. Only 6% of the 100 mothers used the postnatal services of the PHC center. The findings indicate that the provision of upgraded services by itself is insufficient to overcome the lack of health care motivation on the part of the target population.^ieng

Neonatal iron status is impaired by maternal obesity and excessive weight gain during pregnancy.

OBJECTIVE: Maternal iron needs increase sixfold during pregnancy, but obesity interferes with iron absorption. We hypothesized that maternal obesity impairs fetal iron status. STUDY DESIGN: Three hundred and sixteen newborns with risk factors for infantile iron deficiency anemia (IDA) were studied to examine obesity during pregnancy and neonatal iron status. Erythrocyte iron was assessed by cord blood hemoglobin (Hb), zinc protoporphyrin/heme (ZnPP/H) and reticulocyte-ZnPP/H, and storage iron by serum ferritin. RESULT: Women with body mass index (BMI) ⩾ 30 kg m(-)(2), as compared with non-obese women, delivered larger offspring with higher reticulocyte-ZnPP/H and lower serum ferritin concentrations (P<0.05 for both). With increasing BMI, the estimated body iron was relatively lower (mg kg(-)(1)) and the ratio of total Hb-bound iron (mg) per total body iron (mg) increased. Maternal diabetes compromised infant iron status, but multivariate analysis demonstrated that obesity was an independent predictor. CONCLUSION: Obesity during pregnancy and excessive weight gain are independent risk factors for iron deficiency in the newborn.

Extension of the intensive phase reduces relapse but not failure in a regimen with rifampicin throughout.

SETTING: Damien Foundation tuberculosis (TB) control projects in Bangladesh. OBJECTIVE: To assess the effectiveness of extending the intensive phase (P1) of treatment by 1 month for patients who are smear-positive after 2 months of a 6-month regimen containing rifampicin (RMP) throughout. DESIGN: Prospective operational study randomising P1 extension for new smear-positive cases with any number of acid-fast bacilli in the 2-month smear (2M+). Smear-defined failures and relapses underwent culture and drug susceptibility testing in addition to DNA sequencing of the rpoB gene before and after treatment. RESULTS: Of 16,708 patients evaluated, 12,967 were smear-negative at 2 months (2M-); 1871 and 1870 2M+ were randomised to no extension or extension. Respectively 0.3% (95%CI 0.2-0.4), 1.2% (95%CI 0.7-1.8) and 2.0% (95%CI 1.4-2.8) smear- and culture-positive failures, and 1.2% (95%CI 1.0-1.4), 2.6% (95%CI 1.9-3.4) and 0.9% (95%CI 0.5-1.4) relapses were detected. Extension significantly reversed the relative risk (RR) of relapse of 2M+ vs. 2M- patients from 2.2 (95%CI 1.6-3.0) to 0.7 (95%CI 0.4-1.2). The RR for failure remained high, at 7.3 (95%CI 4.7-11.5) with and 4.2 (95%CI 2.5-7.2) without extension. More multi-drug resistance was found after extension, but acquired RMP resistance was similar in all arms. The fair sensitivity of the 2-month smear for failure or relapse (40%) was offset by a very low positive predictive value (3%). CONCLUSIONS: Extension of P1 is very inefficient with this 6-month regimen. Operational research should define appropriate algorithms allowing an earlier switch to the next higher regimen for those in need, using follow-up smears for screening.