First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

Effect of Continuous Administration of Enalapril Maleate on the Oocyte Quality and In Vitro Production of Parthenote Embryos in Nulliparous and Multiparous Goats Undergoing Serial Laparoscopic Ovum Pick-Up.

The aim of this work was to determine the effect of enalapril maleate administration, during oocyte recovery by serial laparoscopic ovum pick-up (LOPU), on the ovarian response and in vitro embryo production (IVP). Twenty cross-bred goats were allocated equally into two groups: Nulliparous and Multiparous. In each group, five animals were selected to receive daily doses of enalapril maleate during the hormonal protocol. Estrus was synchronized by a PGF2α analog, followed 48 h later by insertion of an intravaginal device with progesterone. Forty-eight hours after, a single dose of FSH/eCG was administered. The FSH/eCG doses were repeated three times, on every four day. Oocytes were recovered by LOPU 24 h after each FSH/eCG dose. Viable oocytes were matured in vitro, to be parthenogenetically activated and cultured for 72 h to the cleavage stage. The drug treatment increased the proportion of total follicles observed at LOPU (p < 0.01) in multiparous goats. In both parity groups, enalapril administration had no effect on the proportion or quality of oocytes recovered. Furthermore, the number of embryos cleaved was similar between the groups. Thus, enalapril maleate affected the ovarian response in multiparous animals only and had no effect on the oocyte quality or IVP.

Cryogenic mount for mirror and piezoelectric actuator for an optical cavity.

We present the development of a mount that accommodates a mirror and a piezoelectric actuator with emphasis on physical needs for low temperature operation. The design uses a monolithic construction with flexure features that allow it to steadily hold the mirror and the piezoelectric actuator without glue and accommodate differential thermal contraction. The mount is small and lightweight, adding little heat capacity and inertia. It provides a pre-loading of the piezoelectric actuator as well as a good thermal connection to the mirror and a thermal short across the piezoelectric actuator. The performance of the assemblies has been tested by thermally cycling from room temperature down to 3 K more than a dozen times and over one hundred times to 77 K, without showing any derating. Such mounts are proposed for the cryogenic optical enhancement cavities of the ALPHA experiment at CERN for laser spectroscopy of antihydrogen and for hydrogen spectroscopy in our laboratory at UFRJ.

Multimodal and non-linear optical microscopy applications in reproductive biology.

A plethora of optical techniques is currently available to obtain non-destructive, contactless, real time information with subcellular spatial resolution to observe cell processes. Each technique has its own unique features for imaging and for obtaining certain biological information. However none of the available techniques can be of universal use. For a comprehensive investigation of biological specimens and events, one needs to use a combination of bioimaging methods, often at the same time. Some modern confocal/multiphoton microscopes provide simultaneous fluorescence, fluorescence lifetime imaging, and four-dimensional imaging. Some of them can also easily be adapted for harmonic generation imaging, and to permit cell manipulation technique. In this work we present a multimodal optical workstation that extends a commercially available confocal microscope to include nonlinear/multiphoton microscopy and optical manipulation/stimulation tools. The nonlinear microscopy capabilities were added to the commercial confocal microscope by exploiting all the flexibility offered by the manufacturer. The various capabilities of this workstation as applied directly to reproductive biology are discussed. Microsc. Res. Tech. 79:567-582, 2016. © 2016 Wiley Periodicals, Inc.

Matrix isolation sublimation: An apparatus for producing cryogenic beams of atoms and molecules.

We describe the apparatus to generate cryogenic beams of atoms and molecules based on matrix isolation sublimation. Isolation matrices of Ne and H2 are hosts for atomic and molecular species which are sublimated into vacuum at cryogenic temperatures. The resulting cryogenic beams are used for high-resolution laser spectroscopy. The technique also aims at loading atomic and molecular traps.

Measurement of the hydrodynamic radius of quantum dots by fluorescence correlation spectroscopy excluding blinking.

One of the most important properties of quantum dots (QDs) is their size. Their size will determine optical properties and in a colloidal medium their range of interaction. The most common techniques used to measure QD size are transmission electron microscopy (TEM) and X-ray diffraction. However, these techniques demand the sample to be dried and under a vacuum. This way any hydrodynamic information is excluded and the preparation process may alter even the size of the QDs. Fluorescence correlation spectroscopy (FCS) is an optical technique with single molecule sensitivity capable of extracting the hydrodynamic radius (HR) of the QDs. The main drawback of FCS is the blinking phenomenon that alters the correlation function implicating in a QD apparent size smaller than it really is. In this work, we developed a method to exclude blinking of the FCS and measured the HR of colloidal QDs. We compared our results with TEM images, and the HR obtained by FCS is higher than the radius measured by TEM. We attribute this difference to the cap layer of the QD that cannot be seen in the TEM images.

Source of slow lithium atoms from Ne or H2 matrix isolation sublimation.

We have studied, via laser absorption spectroscopy, the velocity distribution of (7)Li atoms released from cryogenic matrices of solid neon or molecular hydrogen. The Li atoms are implanted into the Ne or H(2) matrices--grown onto a sapphire substrate--by laser ablation of a solid Li or LiH precursor. A heat pulse is then applied to the sapphire substrate sublimating the matrix together with the isolated atoms. With a NiCr film resistor deposited directly onto the sapphire substrate we are able to transfer high instantaneous power to the matrix, thus reaching a fast sublimation regime. In this regime the Li atoms can get entrained in the released matrix gas, and we were also able to achieve matrix sublimation times down to 10 µs for both H(2) or Ne matrix, enabling us to proceed with the trapping of the species of our interest such as atomic hydrogen, lithium, and molecules. The sublimation of the H(2) matrix, with its large center-of-mass velocity, provides evidence for a new regime of one-dimensional thermalization. The laser ablated Li seems to penetrate the H(2) matrix deeper than it does in Ne.

Spectroscopy of lithium atoms sublimated from isolation matrix of solid Ne.

We have studied, via laser absorption spectroscopy, the velocity distribution of (7)Li atoms released from a solid neon matrix at cryogenic temperatures. The Li atoms are implanted into the Ne matrix by laser ablation of a solid Li precursor. A heat pulse is then applied to the sapphire substrate sublimating the matrix together with the isolated atoms at around 12 K. We find interesting differences in the velocity distribution of the released Li atoms from the model developed for our previous experiment with Cr [R. Lambo, C. C. Rodegheri, D. M. Silveira, and C. L. Cesar, Phys. Rev. A 76, 061401(R) (2007)]. This may be due to the sublimation regime, which is at much lower flux for the Li experiment than for the Cr experiment, as well as to the different collisional cross sections between those species to the Ne gas. We find a drift velocity compatible with Li being thermally sublimated at 11-13 K, while the velocity dispersion around this drift velocity is low, around 5-7 K. With a slow sublimation of the matrix we can determine the penetration depth of the laser ablated Li atoms into the Ne matrix, an important information that is not usually available in most matrix isolation spectroscopy setups. The present results with Li, together with the previous results with Cr suggest this to be a general technique for obtaining cryogenic atoms, for spectroscopic studies, as well as for trap loading. The release of the isolated atoms is also a useful tool to study and confirm details of the matrix isolated atoms which are masked or poorly understood in the solid.

Uso de pinças ópticas para avaliação do potencial zeta de hemácias em EDTA, CPD-SAGM e estocadas / Use of optical tweezers for evaluation of szeta potential of red blood cells in EDTA, CPD-SAGM and stored in blood banks

A hemácia é carregada negativamente, principalmente devido ao ácido siálico que gera um potencial elétrico denominado Potencial Zeta que impede a aglutinação intravascular. Os testes de hemaglutinação na rotina transfusional, necessitam de substâncias potencializadoras, das quais muitas agem diminuindo o Potencial Zeta para se ter maior sensibilidade. Através da pinça óptica, ferramenta capaz de capturar células utilizando a luz, foi proposta uma metodologia para quantificar o potencial zeta e aplicar em hemácias coletadas com EDTA e estocadas em CPD-SAGM (visando avaliar alterações de cargas da membrana relacionadas a lesões de armazenamento. Os potenciais zeta em CPD-CAGM foram superiores (-14,8 mV) aos em EDTA (-7,9 mV) e decrescentes a partir do primeiro dia de armazenamento, estabilizando-se a partir da terceira semana com potencial zeta -7,6 mV. Hemácias com CPD-SAGM apresentaram potencial zeta maior, pois possivelmente este conservante evitou lesões mais significativas da membrana que poderiam alterar as cargas. A redução do potencial zeta no armazenamento pode ser consequência de enzimas liberadas de leucócitos lisados que tenham alterado as glicoforinas da membrana. A metodologia permitiu avaliar o potencial zeta em diferentes condições e poderá contribuir na padronização de técnicas de hemaglutinação com diferentes meios potencializadores e no melhor conhecimento das lesões de estocagem para fins transfusionais.

Studying taxis in real time using optical tweezers: applications for Leishmania amazonensis parasites.

Beads trapped by an optical tweezers can be used as a force transducer for measuring forces of the same order of magnitude as typical forces induced by flagellar motion. We used an optical tweezers to study chemotaxis by observing the force response of a flagellated microorganism when placed in a gradient of attractive chemical substances. This report shows such observations for Leishmania amazonensis, responsible for leishmaniasis, a serious disease. We quantified the movement of this protozoan for different gradients of glucose. We were able to observe both the strength and the directionality of the force. The characterization of the chemotaxis of these parasites can help to understand the mechanics of infection and improve the treatments employed for this disease. This methodology can be used to quantitatively study the taxis of any kind of flagellated microorganisms under concentration gradients of different chemical substances, or even other types of variable gradients such as temperature and pressure.

Core-shell CdS/Cd(OH)2 quantum dots: synthesis and bioconjugation to target red cells antigens.

We report a new and efficient methodology of labelling red blood cells, in order to investigate the expression of anti-A antigen, employing luminescent semiconductor nanocrystals. Highly luminescent and stable core-shell cadmium sulphide/cadmium hydroxide [CdS/CdS(OH)2] colloidal particles were obtained in the nanometre size range. The surface of these particles was characterized by using a monoclonal anti-A antibody via a one-step glutaraldehyde cross-linking procedure, followed by conjugation of the particles to red cells of blood groups A+, and O+. Laser scanning confocal microscopy images indicated that after conjugation for 30 min, A+ and erythrocytes presented different patterns of dual bright emission whereas the O+ group cells showed no emission. We suggest that this labelling procedure may be applied as a quantitative tool to investigate the distribution and expression of alloantigen in red blood cells.

Raman, hyper-Raman, hyper-Rayleigh, two-photon luminescence and morphology-dependent resonance modes in a single optical tweezers system.

We present a setup of optical tweezers combined with linear and nonlinear microspectroscopies that enhances the capabilities of capture and analysis of both techniques. We can use either a continuous-wave (cw) Ti:sapphire laser for Raman measurements or a pulsed femtosecond Ti:sapphire laser that permitted the observation of nonlinear results such as hyper-Raman, hyper-Rayleigh, and two-photon luminescence. Only the high peak intensity of the femtosecond laser allows the observation of all these nonlinear spectroscopies. The sensitivity of our system also permitted the observation of morphology-dependent resonance (MDR) modes of a single stained trapped microsphere of 6 microm. The possibility of performing spectroscopy in a living microorganism optically trapped in any desired neighborhood would mean that one can dynamically observe the chemical reactions and/or mechanical properties changing in real time.

Optical tweezers for measuring red blood cell elasticity: application to the study of drug response in sickle cell disease.

The deformability of erythrocytes is a critical determinant of blood flow in microcirculation. By capturing red blood cells (RBC) with optical tweezers and dragging them through a viscous fluid we were able to measure their overall elasticity. We measured, and compared, the RBC deformability of 15 homozygous patients (HbSS) including five patients taking hydroxyurea (HU) for at least 6 months (HbSS/HU), 10 subjects with sickle cell trait (HbAS) and 35 normal controls. Our results showed that the RBC deformability was significantly lower in haemoglobin S (HbS) subjects (HbSS and HbAS), except for HbSS/HU cells, whose deformability was similar to the normal controls. Our data showed that the laser optical tweezers technique is able to detect differences in HbS RBC from subjects taking HU, and to differentiate RBC from normal controls and HbAS, indicating that this is a very sensitive method and can be applied for detection of drug-response in sickle cell disease.

Elastic properties of irradiated RBCs measured by optical tweezers.

BACKGROUND: Gamma irradiation of RBCs results in the production of reactive oxygen capable of initiating the process of membrane lipid peroxidation and accelerates the leakage of potassium ions from RBCs, resulting in an increase of internal viscosity. STUDY DESIGN AND METHODS: The elastic properties of irradiated and stored RBC units were studied using laser optical tweezers. The laser trapped the cell and the membrane elasticity was analyzed, measuring the cell deformation in six different drag velocities. Five RBC units were split into two portions. One portion received a gamma irradiation dose of 25 Gy, and the second one was used as a control and was not irradiated. All units were stored (4 degrees C), and the elasticity was examined on Days 1, 14, 21, and 28. RESULTS: Elastic properties (mu) from irradiated RBCs stored for 21 and 28 days were significantly affected compared with control cells (21 days: control, 0.3 +/- 0.03 x 10(-3); irradiated, 3.5 +/- 1.3 x 10(-3) dyn/cm; p < 0.001; and 28 days: control, 0.5 +/- 0.09 x 10(-3); irradiated, 14 +/- 3.2 x 10(-3) dyn/cm; p < 0.001). CONCLUSION: The sensitivity of the laser optical tweezers method showed that there is no significant change in elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not. However, beyond 21 days of storage, irradiated units demonstrate decreased elasticity.

Effects of environmental and organizational factors on the health of shiftworkers of a printing company.

This study explored the effects of environmental and organizational stressors on the health of shiftworkers in a printing company (n = 124). A questionnaire was used to gather data on work history, organizational factors, psychosocial characteristics, medical history, present health, occupational and non-occupational exposures, and lifestyle factors. The perception of environmental and organizational conditions was associated (P < 0.05) with chronic back pain (odds ratio [OR], 1.29), varicose veins (OR, 1.35), allergic rhinitis (OR, 1.27), depression (OR, 1.45), and gastritis (OR, 1.15). Anxiety scores were associated with allergic rhinitis (OR, 1.14) and skin allergy (OR, 1.09). Shiftwork was a significantly risk factor for conjunctivitis (OR, 3.68), depression (OR, 0.23), cardiac arrhythmia (OR, 7.13), and gastritis (OR, 4.38). Other associations included tenure and chronic back pain (OR, 4.89), toluene exposure and skin allergy (OR, 3.76), worksite and conjunctivitis (OR, 7.0), and worksite and dermatitis (OR, 1.24 to 4.95). The number of hours of exercise per week was associated with varicose veins (OR, 4.33), and alcohol intake was associated with cardiac arrhythmia (OR, 6.74).

[Training of local health managers: process of identification of strategic actions]. / Formação de gestores locais de saúde: processos para identificar estratégias de atuação.

In Brazil, in recent years, as a result of the increasing participation of county authorities in health care, a need for tools which would contribute to the better preparation of local administrators, complementary to the activities of a more academic nature, has been recognized. One of the possible alternatives is the exploitation of experiences, regarded as successful, in local health care planning and administration, by using them as material for "case studies" in activities with selected groups of health care administrators thus, stimulating the identification of those elements which contributed to the favorable results attained, and their interactions, in the quest for analogies which would facilitate the identification of new perspectives for their own situations. In this article an experience of a "case studies" development, in response to a demand from UNICEF, based on 8 counties in from the North and Northeast, which were successful in using a "focus approach" in their organization of data and their utilisation in a seminar with 21 local managers from both regions, is presented. During the seminar the local health managers attained greater knowledge of the strategies implemented and identified feasible intervention alternatives. The methodological proposition of teaching on the basis of case studies, using a conceptual strategy of grouping experiences according to specific dimensions enabled local health managers to learn from their practical experiences.

[Referred morbidity and the utilization of health services in Brazilian urban areas: methodology]. / Morbidade referida e utilização de serviços de saúde em localidades urbanas brasileiras: metodologia.

The reorganization of the Brazilian health system in the last few years has been based on the decentralization of the services and the integration of preventive and curative medicine, then creating new needs in term of the health information needed for health planning. The planning and administration of a regionalized and integrated health system calls for information about the population's morbidity profile, and the description of the pattern of the utilization of the health services, which can be obtained by means of household health surveys. The methodology utilized in a household health survey carried out in the Metropolitan Area of S. Paulo, Brazil, from July 1989 to June 1990, is described. This survey has some distinct methodological characteristics, such as the sampling process that defined the domains by age and sex, allowing an analysis of populational groups less well represented in the general population, as that of less than one year of age and that of aged people, as well as the use of the results of the latest Brazilian Census in 1991 for adjusting the sample.

[Risk factors associated with infant mortality in 2 areas of the metropolitan region of São Paulo (Brazil), 1984-1985. Proposal for predictive instruments]. / Fatores de risco associados à mortalidade infantil em duas áreas da região metropolitana de São Paulo (Brasil), 1984-1985. Proposta de instrumentos preditivos.

The application of the concept of "risk" to maternal and child health was studied, the starting point being the World Health Organization's proposal for a "risk approach" in health services. The study was concerned with the development of a scoring system for the identification of groups at high risk of death during the first year of life, whether in the neonatal or the post-neonatal periods, and was based on a case-control study. The case group consists of children under one year of life, whose deaths had been registered during the years of 1984 and 1985 and whose parents lived in Cotia and Vargem Grande Paulista (149 cases). The control group was a probabilistic sample of 216 children born in 1984, which survived the first year of life and whose parents lived in the same area. All the mothers were interviewed by means of a questionnaire for the identification of independent variables associated with infant death. The statistically associated variables were grouped in four scales: to be used, respectively, in the antenatal period, prior to delivery and in the neonatal and post-neonatal periods. The variables were weighted in the scales according to the values of the odds ratio, and they have different cut points, each one having its own sensibility, specificity and predictive value.

Diversity in provision and utilization of maternal and child health care in an urban area of Brazil.

Health services in Brazil are characterized by a multiplicity of providers. While many are ultimately funded from public sources, notably the National Social Security Scheme, a large proportion are provided by the private sector. This variety of providers of health care has hindered the development of comprehensive care and the coverage of those in greatest need. In recent years increasing attention has been given to ways of reducing the undesirable aspects of this situation. The Federal and State Governments have established committees to improve coordination between services and set up several pilot programmes. One concerned with improving the effective coverage of maternal and child services has been established in Sorocaba. Sorocaba is a medium-sized city which is typical of the many urban centres in the state of São Paulo. This paper describes a study which has provided information from a representative sample of women, who had recently had a baby, on their eligibility for care from the various health services available in the city and on their actual use of those services. This information has been used in the implementation of a pilot programme. Most mothers attended some service for antenatal care. Almost 20% used several services because their entitlement varied from scheme to scheme and some services provided only specific components of care. This applied also to preventive and curative infant care.

PIP: A survey of 296 mothers, with children born in March 1979, was conducted in Sorocaba, Sao Paulo State, Brazil, to describe the use and payment of health care, for baseline data for a maternal-child health program. Sorocaba, 96 km northwest of Sao Paulo, has 260,000 people and an infant mortality of 70/1000. Health care is dominated by private physicians and hospitals, and is largely reimbursed by public sources through the INAMPS social security payroll taxes, federal, state and municipal funds. There are also organized physician co-ops, and prepaid health plans. Because of diversity funding and services, there is an emphasis on curative medicine, duplication, and inequity of care. Extensive information is included in tables on socioeconomic, education, medical, insurance coverage, type of care received and residence characteristics of the subjects, expressed as percentages. Most (83%) mothers belonged to the Federal Social Security plan, and a third also belonged to a private insurance plan, usually through the husband's employment. Only 4% were not covered, except State and Municipal providers. Only 1.7% had no prenatal care. Any drugs needed were paid in part, although 17% had to go to more than one provider to get them. 98% were delivered in hospitals, 30% by Cesarean section, for which doctors receive higher fees. Many (62%) took their children for care by 2 months of age. Equal numbers used private or state health centers, while 14% used several sources. For infants' curative services 56% used private physicians or hospitals, 17% paid for care and 86% paid for drugs. Women in lower socioeconomic groups or with less education began prenatal care later and made significantly fewer visits for prenatal or infant care (p.001). The study has prompted discussions on coordination of outpatient services and identification of high risk patients.