Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program Would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n = 5; receiving 500 mg of bST in regular intervals) and control group (n = 5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (> 3 mm in diameter; 12.2 compared with 8.7: p, < 0.05) and of small antral follicles (< 5 mm; 9.1 compared with 6.5; p < 0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p = 0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p = 0.07), bST showed no effect on cleavage and blastocyst production rates (p > 0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p < 0.001) as well as the number of follicles aspirated (p < 0.001), and oocytes recovered (p < 0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs. (c) 2008 Elsevier B.V. All rights reserved.

First polymorphisms in JY-1 gene in cattle (Bos taurus indicus) and their association with sexual precocity and growth traits

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Produção in vitro de embriões bovinos: utilização de diferentes fontes de gonadotrofinas na maturação dos oócitos

O presente trabalho foi conduzido com o objetivo de avaliar o efeito da utilização de diferentes fontes de gonadotrofinas para maturação in vitro dos oócitos bovinos fecundados e desenvolvidos in vitro sobre as taxas de clivagem (TC) e de blastocistos (TBL). Oócitos imaturos provenientes de ovários de vacas de abatedouro foram submetidos a maturação in vitro sob diferentes condições: meio TCM 199, acrescido de 10% de soro de vaca em estro (SVE), aditivos, hepes, NaHCO3, piruvato de sódio, antibióticos (meio B-199), 20 UI/mL de PMSG e 10 UI/mL de hCG (PMSG/hCG) ou meio B-199, acrescido de 5 mig/mL de FSH e 5 mig/mL de LH (FSH/LH). Seguidos 24 h de cultura a 38,5ºC em atmosfera com 5% de CO2, os oócitos maturos foram incubados com sêmen descongelado durante 18 a 21 horas. Após esse período, os oócitos foram transferidos para placas contendo microgotas de meio Ménezo suplementado com 10% de SVE e células epiteliais do oviduto bovino em suspensão, cobertas com óleo de silicone, os quais permaneceram em cultura por mais 9 dias. Os dados foram analisados pelo teste do Qui-quadrado. A TC e a TBL, para PMSG/hCG e FSH/LH, foram 60 e 13,9% e 61,2 e 10,6%, respectivamente. Não houve diferença entre os tratamentos com relação a TC ou a TBL. Esses resultados sugerem que ambas as fontes de gonadotrofinas podem ser utilizadas para maturação in vitro dos oócitos fecundados e desenvolvidos in vitro.

Study of the interaction between cardiolipin bilayers and methylene blue in polymer-based Layer-by-Layer and Langmuir films applied as membrane mimetic systems

An increase of the reports involving mimetic systems has been observed. Briefly, these systems use biological phospholipids to exploit specific interactions between membrane-models and drugs. Here, the Layer-by-Layer (LbL) and Langmuir techniques were used to investigate the interaction between cardiolipin (CLP-negative phospholipid) and a cationic-like drug methylene blue (MB). Supported by a cationic polyelectrolyte (PAH), LbL films containing PAH/(CLP + MB) and PAH/(CLP + MB + AgNP) were grown up to 14 bilayers. The optical microscopy analysis revealed a decrease of the CLP vesicle sizes in the presence of MB as a possible consequence of the MB action onto the mechanical properties of the CLP membrane. From FTIR spectra, changes mainly related to peak position and band intensity and shape were observed in the spectra from PAH/CLP when in the presence of MB. The latter supports that the interactions between the phosphate and amine charged groups from CLP and PAH, respectively, established during the LbL film fabrication, besides the CLP hydrocarbon environment, are influenced by the presence of MB. Using the micro-Raman technique, a chemical mapping was build based on MB spectrum by resonance Raman scattering (RRS) and surface-enhanced resonance Raman scattering (SERRS). The later phenomenon was activated by Ag nanoparticles (AgNPs) trapped within the LbL film allowing collecting spectra for a single bilayer of PAH/(CLP + MB + AgNP). A rough estimation showed a SERRS amplification of 10(3) in comparison to RRS spectra. As a complementary approach, Langmuir films of CLP in the presence of co-spread MB were investigated through surface pressure vs mean molecular area (pi-A) isotherms. The results showed that for concentrations of MB below 100 mol%, the drug is expelled to water subphase for high values of surface pressure (condensed phase). For concentration at 100% and higher, the MB keeps bound to CLP floating monolayer. (C) 2010 Elsevier B.V. All rights reserved.

Effect of vesicles of dimethyldioctadecylammonium chloride and phospholipids on the rate of decarboxylation of 6-nitrobenzisoxazole-3-carboxylate

Sonicated mixtures of dimethyldioctadecylammonium chloride (DODAC), egg phosphatidylcholine (PC), dimyristoyl phosphatidylcholine (DMPC), and dipalmitoyl phosphatidylcholine (DPPC) were used to analyze vesicle effects on the rate of decarboxylation of 6-nitrobenzisoxazol-3-carboxylic acid (Nboc). Electron microscopic images of the vesicles were obtained with trehalose, a know cryoprotector. Phase diagrams and phase transitions temperatures of the vesicle bilayers were determined. Nboc decarboxylation rates increased in the presence of vesicles prepared with both phospholipids and DODAC/phospholipid mixtures. Quantitative analysis of vesicular effects was done using pseudophase models. Phospholipids catalyzed up to 140-fold while the maximum catalysis by DODAC/lipid vesicles reached 800-fold. Acceleration depends on alkyl chain length, fatty acid insaturation of the lipids, and the DODAC/phospholipid molar ratio. Catalysis is not related to the liquid crystalline-gel state of the bilayer and may be related to the relative position of Nboc with respect to the interface.

Structural analysis of fertilization in the fish Brycon orbignyanus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Attitude propagation using non-singular canonical variables

Three sets of non-singular canonical variables for the rotational motion are analyzed. These sets are useful when the angle between z-axis of a coordinate system fixed in artificial satellite ( here defined by the directions of principal moments of inertia of the satellite) and the rotational angular momentum vector is zero or when the angle between Z-inertial axis and rotational angular momentum vector is zero. The goal of this paper is to compare all these sets and to determine the benefits of their uses. With this objective, the dynamical equations of each set were derived, when mean hamiltonian associate with the gravity gradient torque is included. For the torque-free rotational motion, analytical solutions are computed for symmetrical satellite for each set of variables. When the gravity gradient torque is included, an analytical solution is shown for one of the sets and a numerical solution is obtained for one of the other sets. By this analysis we can conclude that: the dynamical equation for the first set is simple but it has neither clear geometrical nor physical meaning; the other sets have geometrical and physical meaning but their dynamical equations are more complex.

Effects of aspirin on mesenteric lymph nodes of rabbits as basis for its use on lymph nodes metastases

OBJETIVO: Avaliar os efeitos do ácido acetilsalicílico a 10% e 20% em linfonodos mesentéricos de coelhos para posterior embasamento e uso em metástases linfonodais. MÉTODOS: Um total de 20 linfonodos de 20 coelhos (divididos aleatoriamente em quatro grupos) foi avaliado. As soluções de aspirina a 10% (grupos A e C) e 20% (grupos B e D) foram injetadas em linfonodos mesentéricos de coelhos sadios e seus efeitos macroscópicos e histológicos foram avaliados em 24 horas (grupos A e B) e em sete dias (grupos C e D). RESULTADOS: Nos grupos avaliados em 24 horas (A e B) foi verificada intensa necrose e hemorragia, aumento importante de apoptose em todo o linfonodo, com alargamento dos seios medulares e aumento dos centros germinativos. Nos grupos avaliados em sete dias (C e D) também houve aumento da apoptose, com maior elevação de histiócitos e diminuição importante da necrose; a hemorragia foi ausente e aumento de células gigantes foi visualizado, conferindo processo inflamatório crônico do tipo corpo estranho. Não houve diferença entre as concentrações utilizadas (10 e 20%) em nenhuma das comparações. CONCLUSÕES: A injeção de aspirina em linfonodos causou necrose e um aumento de apoptose após 24 horas e após sete dias de tratamento, houve regeneração dos gânglios linfáticos, com diminuição intensa de necrose e grande aumento de apoptose. Uma vez que o aumento de apoptose é um dos pilares dos tratamentos antineoplásicos, estes resultados experimentais embasam eventual aplicação clínica da aspirina no tratamento de metástases linfonodais.

Recombinant luteinizing hormone supplementation to recombinant follicle-stimulation hormone during induced ovarian stimulation in the GnRH-agonist protocol: A meta-analysis

Purpose: to compare the efficacy of recombinant LH supplementation for controlled ovarian stimulation in recombinant FSH and GnRH-agonist protocol.Methods: Search strategies included on-line surveys of databases. The fixed effects model was used for odds ratio and effect size (weighted mean difference). Four trials fulfilled the inclusion criteria.Results: a fewer days of stimulation (p < 0.0001), a fewer total amount of r-FSH administered (p < 0.0001) and a higher serum estradiol levels on the day of hCG administration (p < 0.0001) were observed for the r-LH supplementation protocol. However, differences were not observed in number of oocyte retrieved, number of mature oocytes, clinical pregnancy per oocyte retrieval, implantation and miscarriage rates.Conclusions: more randomized controlled trials are necessary before evidence-based recommendations regarding exogenous LH supplementation in ovarian stimulation protocols with FSH and GnRH-agonist for assisted reproduction treatment can be provided.

Administration of beta(2)-adrenergic agonists during the peri-implantation period does not improve implantation or pregnancy rates in intracytoplasmic sperm injection (ICSI) cycles

Purpose: the objective of the present investigation was to determine implantation and pregnancy rates in patients undergoing ICSI and treated with beta(2)-adrenergic agonists, considering the uterine-relaxing action of these agents.Methods: A total of 225 women undergoing ICSI at the Center for Human Reproduction, Sinha Junqueira Maternity Foundation, entered the study. Patient participation in each group was random, by drawing lots, using a randomization table previously elaborated for the study (2:2:1). The group I (90 women) received 10 mg of terbutaline daily for 15 days starting on the day of oocyte retrieval; group II (90 women) received 20 mg of ritodrine daily during the same period of time as group I; group III (45 patients) received no treatment and was used as control. The evaluation was interrupted in 3 patients of group I and in 30 patients of group II because of a high incidence of side effects.Results: Pregnancy, implantation, and miscarriage rates were not significantly different (p>0.05) between the three groups: 29.88%, 13.25%, and 26.9% for group I; 33.33%, 17.5%, and 10.0% for group II; 28.88%, 15.07%, and 15.38% for group III, respectively.Conclusions: the results of this study do not support the routine use of beta(2)-adrenergic agonists during the peri-implantation period in assisted reproductive technology cycles.

Comparison of day 2 embryo quality after conventional ICSI versus intracytoplasmic morphologically selected sperm injection (IMSI) using sibling oocytes

Objective: To evaluate whether intracytoplasmic morphologically selected sperm injection (IMSI) could influence early paternal effects by observing embryo quality at day 2.Study design: The study included 30 couples with at least one of the following criteria: male factor infertility, at least 2 previous failures of implantation or previous miscarriages after IVF/ICSI. Sibling oocytes of each patient were randomly assigned to either the ICSI group or the IMSI group. For IMSI, spermatozoa were selected at 8400x magnification through an inverted microscope equipped with Nomarski differential interference contrast optics, Uplan Apo 100x oil/1.35 objective lens and variable zoom lens. For conventional ICSI, spermatozoa were selected at 400x magnification. An embryo was defined as top quality if there were four identical blastomeres on day 2 with no fragments or multinucleation of blastomeres. Data were analysed using the Wilcoxon and chi-squared tests. The significance level was set at P < 0.05. The variables were analysed in relation to the general population and the subpopulations with or without male factor.Results: A total of 331 MII oocytes (30 oocyte retrievals) were selected and injected by the ICSI (n: 172) or IMSI (n: 159) procedure. For IMSI, only spermatozoa classified as morphologically normal at high magnification were used. No differences (P > 0.05) in fertilisation rate (ICSI: 70.9%; IMSI: 70.4%), early embryo cleavage rate (ICSI: 66.9%; IMSI: 60.4%) or cleavage rate (ICSI: 99.2%; IMSI: 99.1%) were observed. on day 2, as compared to ICSI, IMSI provided a similar proportion of top quality embryos (ICSI: 57.8%; IMSI: 52.2%; P > 0.05). These results were not influenced by the presence or absence of male factor.Conclusion: In terms of embryo quality at day 2, IMSI had the same performance as conventional ICSI. However, we cannot exclude the possibility that IMSI effects occur only as a positive later paternal effect. (C) 2010 Elsevier B.V. All rights reserved.

GnRH agonist versus GnRH antagonist in IVF/ICSI cycles with recombinant LH supplementation: DNA fragmentation and apoptosis in granulosa cells

Objective: To compare the level of apoptosis and DNA fragmentation in the human granulosa cell (GC) layer exposed to an agonist or antagonist of GnRH in intracytoplasmic sperm injection (ICSI) cycles supplemented with recombinant LH (rLH).Study design: Patients without ovulatory dysfunction, aged <= 37 years and in their first ICSI cycle were prospectively randomised to receive either a long GnRH agonist protocol or a multi-dose antagonist protocol. In both groups, recombinant FSH supplemented with rLH was used for ovarian stimulation, and the GCs were collected during oocyte denudation. The GCs were then analysed for DNA fragmentation by TUNEL assay and for apoptosis using the annexin-V assay. The outcomes were given as the percentage of GCs with DNA fragmentation and apoptosis out of the total number of GCs analysed. Comparison of the agonist versus the antagonist group was performed using the Mann-Whitney test.Results: DNA fragmentation: 32 patients were included in either the GnRH agonist group (n = 16) or the antagonist group (n = 16). The percentage of GCs with positive DNA fragmentation did not differ significantly (P = 0.76) between the agonist group (15.5 +/- 9.4%) and the antagonist group (18.8 +/- 13.3%). Apoptosis: 28 patients were included in either the GnRH agonist group (n = 14) or the antagonist group (n = 14). The percentage of GCs positive for apoptosis did not differ significantly (P = 0.78) between the agonist group (34.6 +/- 14.7%) and the antagonist group (36.5 +/- 22%).Conclusions: The results suggest that therapy with either an agonist or antagonist of GnRH is associated with comparable levels of DNA fragmentation and apoptosis in granulosa cells in ICSI cycles supplemented with rLH. (C) 2012 Elsevier B.V. All rights reserved.

Seasonal variability in the fertilization rate of women undergoing assisted reproduction treatments

The objective of this study was to evaluate whether seasonality affects human-assisted reproduction treatment outcomes. For this, 1932 patients undergoing intracytoplasmic sperm injection (ICSI) were assigned to a season group according to the day of oocyte retrieval: winter (n = 435), spring (n = 444), summer (n = 469) or autumn (n = 584). Analysis of variance was used to compare the ICSI outcomes. The fertilization rate was increased during the spring (winter: 67.9%, spring: 73.5%, summer: 68.7% and autumn: 69.0%; p < 0.01). In fact, a nearly 50% increase in the fertilization rate during the spring was observed (odds ratio 1.45, confidence interval 1.20-1.75; p < 0.01). The oestradiol concentration per number of oocytes was significantly higher during the spring (winter: 235.8 pg/mL, spring: 282.1 pg/mL, summer: 226.1 pg/mL and autumn: 228.7 pg/mL; p = 0.030). This study demonstrates a seasonal variability in fertilization after ICSI, where fertilization is higher during the spring than at any other time.

Evaluation of N-acetilglucosaminidase and myeloperoxidase activity in patients with endometriosis-related infertility undergoing intracytoplasmic sperm injection

Aim: Inflammation is as an important factor in ovulation with the active participation of leucocytes and their inflammatory mediators. The present study was performed to compare the activity of the inflammatory enzymes myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) in patients with endometriosis-related infertility and in normally ovulating women undergoing intracytoplasmic sperm injection (ICSI).Material and Methods: This prospective study included infertile women undergoing ICSI treatment. These women were divided into two groups: endometriosis anovulation (n = 18) and normally ovulating (n = 20). NAG and MPO activity was evaluated colorimetrically in serum and in follicular fluids obtained at the time of oocyte retrieval.Results: There was a significant correlation between the serum and follicular fluid activities of NAG and MPO (tau = 0.256, P = 0.025; and tau = -0.234, P = 0.041; respectively). Both serum and follicular fluid NAG activities were higher in patients with endometriosis compared to the control group (P < 0.001). MPO follicular fluid activity was lower in patients with endometriosis compared to normally ovulating women (P = 0.016).Conclusion: Infertile patients with endometriosis show a distinct pattern of serum and follicular fluid macrophage/neutrophil activation compared to normally ovulating women undergoing ICSI, which may reflect the role of immune and inflammatory alterations in endometriosis-related infertility.

Relationship between visualization of meiotic spindle in human oocytes and ICSI outcomes: a meta-analysis

The objective of this meta-analysis was to investigate the influence of meiotic spindle visualization in human oocytes on intracytoplasmic sperm injection (ICSI) outcomes. Search strategies included on-line Surveys of databases (MEDLINE, em BASE, Science Citation Index, Cochrane Controlled Trials Register and Ovid). The fixed effect was used for odds ratio. Ten trials fulfilled the inclusion criteria comparing in-vitro and clinical ICSI outcomes with or without visualization of meiotic spindle in fresh and in-vivo matured oocytes. According to the meta-analysis, the results showed statistically significant higher fertilization rate (P < 0.0001) when the meiotic spindle was viewed than when it was not. Moreover, the percentage of pro-nuclear-stage embyros with good morphology (P = 0.003), cleavage rate (P < 0.0001), percentage of day-3 top-quality embryos (P = 0.003) and percentage of embryos that reached the blastocyst stage (P < 0.0001) were statistically significantly better among, embryos derived from oocytes in which meiotic spindle was viewed compared with those in which meiotic spindle was not observed. However, these differences were not observed in the clinical pregnancy or implantation rates. This observation has clinical relevance mainly in countries where there is a legal limit on the number of oocytes to be fertilized. However, additional controlled trials are needed to further confirm these results.