Dietas com nitrogênio não-proteico para fêmeas bovinas superovuladas sem prévia adaptação durante curto tempo e em diferentes fases do ciclo estral

Objetivou-se neste estudo avaliar o efeito do fornecimento, sem prévia adaptação, durante curto tempo e em diferentes fases do ciclo estral, de dietas contendo nitrogênio não-proteico (NNP) na produção, na qualidade e no grau de desenvolvimento de embriões recuperados em fêmeas bovinas superovuladas. Sessenta e oito vacas Nelore foram distribuídas em três grupos: um controle e dois com fornecimento de ureia antes (UA = do dia -5 ao dia 0) e após (UD = dia 0 ao dia 5) a inseminação artificial. As vacas foram mantidas em pastagem e receberam concentrado (3,0 kg/animal/dia) durante 16 dias. Foram formulados dois concentrados, e as dietas totais (concentrado e consumo estimado de pastagem) apresentaram 12,0% (dieta controle) e 14,6% (dieta NNP) de proteína bruta (PB). As vacas foram sincronizadas, superovuladas e inseminadas. Sete dias (dia 7) após a inseminação (dia 0), realizou-se a colheita e análise dos embriões. Amostras de sangue foram coletadas nos dias -5, 0 e 5 para determinação da concentração de nitrogênio ureico plasmático (NUP), glicose, insulina e progesterona. A época de fornecimento de ureia influenciou as concentrações médias de NUP nos dias -5, 0 e 5, mas não tiveram efeito nas concentrações de glicose, insulina e progesterona. O período de fornecimento da ureia teve efeito na porcentagem de mórulas compactas em relação ao total de estruturas (UA = 51,4 vs. UD = 15,3%), em relação ao total de oócitos fecundados (UA = 62,5 vs. UD = 30,6%) e em relação ao total de embriões viáveis (UA = 68,8 vs. UD = 38,6%). No grupo que recebeu ureia depois da inseminação artificial, houve redução de 70,2% da proporção de mórulas compactas por total de estruturas em relação ao grupo que recebeu ureia antes da inseminação. Dietas com nitrogênio não-proteico fornecidas imediatamente após a inseminação promovem aceleração do desenvolvimento embrionário inicial.

Produção in vitro de embriões de ovinos: uma visão crítica do método e de seu resultado a campo

A metodologia de produção in vitro de embriões de ovinos implica no desenvolvimento de meios de maturação, fertilização e cultivo que permitam aumentar a taxa de clivagem e desenvolvimento, tanto para o investimento biotecnológico em programas comerciais, quanto para sua utilização em clonagem e transgenia dessa espécie animal. Do ponto de vista da pesquisa, os ovócitos podem ser obtidos pelas técnicas de punção e slicing a partir de ovários oriundos de matadouros, ou através de aspiração folicular por laparoscopia. Como vantagem, este método permite o uso de uma mesma doadora estimulada hormonialmente em intervalos periódicos, mantida sob rigoroso controle sanitário, o que é de vital importância para a produção de biofármacos em programas que utilisem os ovinos como modelo biológico. Por outro lado, em nosso país a demanda pela multiplicação de animais de alto valor genético, seja pela produtividade ou pelo elevado valor comercial dos mesmos, impõe o desenvolvimento, adaptação e otimização das diferentes metodologias desenvolvidas ao longo dos ultimos anos em laboratórios de referência mundiais. Nesse contexto, cresce de importância o perfeito conhecimento da fisiologia dessa espécie e das raças criadas em nosso país, e da problemática da produção in vitro de seus embriões. Respeitando essas premissas, gerar o desenvolvimento de protocolos que permitam não apenas aumentar a população de ovócitos passíveis de maturação in vitro, mas de sua competência ao desenvolvimento ao estágio de blastocisto, ou, alternativamente, sua transferência a receptoras em estágios precoces do desenvolvimento, evitando assim as conhecidas perdas durante o desenvolvimento in vitro, e o baixo percentual de gestações que chegam a termo, com cordeiro saudáveis. Trata-se de um desafio, que já apresenta os primeiros resultados em nosso país, tanto na produção comercial de embriões produzidos in vitro, quanto em programas de clonagem e transgenia.

Avanços em reprodução assistida

Buscou-se, por meio de uma revisão bibliográfica de publicações científicas, atualizar conhecimentos produzidos sobre reprodução assistida. Inicialmente, investigou-se o histórico e, em seguida, foram apresentados conceitos relativos à infertilidade e sobre as técnicas de reprodução assistida, especificando-se ainda alguns temas relacionados a aspectos obstétricos, epidemiológicos e perinatais. Vários artigos que abordam o tema foram apresentados. Foi discutida a situação no Brasil bem como os vários aspectos assinalados

INTERPOPULATION COMPARISON OF REPRODUCTION OF THE ATLANTIC SHRIMP HIPPOLYTE OBLIQUIMANUS (CARIDEA: HIPPOLYTIDAE)

The genus Hippolyte is represented by typically small shrimps with intriguing mechanisms of reproduction. In order to study possible variability in reproductive aspects among different populations, we conducted an exhaustive comparative study of H. obliquimanus from South (Brazil) and Central American (Costa Rica) waters. The study focuses on fecundity and reproductive output. Mean size of ovigerous females was significantly larger, and both mean reproductive output and mean fecundity were significantly higher in specimens from Costa Rica then in those collected in Brazil. Embryo volume was significantly smaller in the Costa Rican population, and in both populations embryos doubled their volume during embryogenesis. We discuss and compare our findings with the information available regarding H. obliquimanus and other hippolytid shrimp. The reproductive traits of both populations of H. obliquimanus show some important differences which may reflect adaptations to local environmental conditions, demonstrating a high plasticity of reproductive features of the species in Brazilian and Costa Rican waters.

Endometrial claudin-4 and leukemia inhibitory factor are associated with assisted reproduction outcome

Background: Claudin-4 (CLDN4) is one of several proteins that act as molecular mediators of embryo implantation. Recently, we examined immunolabeling of leukemia inhibitory factor (LIF) in the endometrial tissue of 52 IVF patients, and found that LIF staining intensity was strongly correlated with successful pregnancy initiation. In the same set of patients, we have now examined endometrial CLDN4 expression, to see how expression intensity may vary with LIF. We examined CLDN4 in the luteal phase of the menstrual cycle, immediately preceding IVF treatment. Our aim was to compare expression of LIF and CLDN4 in the luteal phase, and document these patterns as putative biomarkers for pregnancy. Methods: Endometrial tissue was collected from women undergoing IVF. Endometrial biopsies were obtained during the luteal phase preceding IVF, and were then used for tissue microarray (TMA) immunolabeling of CLDN4. Previously published LIF expression data were then combined with CLDN4 expression data, to determine CLDN4/LIF expression patterns. Associations between successful pregnancy after IVF and combined CLDN4/LIF expression patterns were evaluated. Results: Four patterns of immunolabeling were observed in the endometrial samples: 16% showed weak CLDN4 and strong LIF (CLDN4(-)/LIF(+)); 20% showed strong CLDN4 and strong LIF (LIF(+)/CLDN4(+)); 28% showed strong CLDN4 and weak LIF (CLDN4(+)/LIF(-)); and 36% showed weak CLDN4 and weak LIF (CLDN4(-)/LIF(-)). Successful implantation after IVF was associated with CLDN4(-)/LIF(+)(p = 0.003). Patients showing this endometrial CLDN4(-)/LIF(+) immunolabeling were also 6 times more likely to achieve pregnancy than patients with endometrial CLDN4(+)/LIF(-) immunolabeling (p = 0.007). Conclusion: The combined immunolabeling expression of CLDN4(-)/LIF(+) in endometrial tissue is a potential biomarker for predicting successful pregnancy in IVF candidates.

Decreased fertility in poor responder women is not related to oocyte morphological status

Introduction: In women showing impaired fertility, a decreased response to ovarian stimulation is a major problem, limiting the number of oocytes to be used for assisted reproduction techniques (ART). Despite the several definitions of poor response, it is still a matter of debate whether young poor responder patients also show a decrease in oocyte quality. The objective in this study was to investigate whether poor ovarian response to the superstimulation protocol is accompanied by impaired oocyte quality. Material and methods: This study included 313 patients younger than 35 years old, undergoing intracytoplasmic sperm injection. Patients with four or fewer MII oocytes (poor-responder group, PR, n = 57) were age-matched with normoresponder patients (NR, n = 256). Results: A higher rate of oocyte retrieval and a trend towards an increase in MII oocyte rate were observed in the NR group when compared to the PR group (71.6 +/- 1.1% and 74.1 +/- 1.0% vs. 56.3 +/- 2.9% and 66.5 +/- 3.7%; p < 0.0001 and p = 0.056, respectively). A trend toward increased implantation rates was observed in the NR group when compared to the PR group (44 and 24.5 +/- 2.0% vs. 28.8 and 16.4 +/- 3.9%; p = 0.0305 and p = 0.0651, respectively). Conclusions: Low response to ovarian stimulation is apparently not related to impaired oocyte quality. However, embryos produced from poor responder oocytes show impaired capacity to implant and to carry a pregnancy to term.

Recoleta uterina como estratégia para aumentar a taxa de embriões em fêmeas bovinas de corte e leite

Embryo recovery rate in superovulated cows after uterine flushing is lower than the ovulation rate, which contributes to the relative inefficiency of the conventional cervical recovery procedure. A simple and easy alternative to improve embryo recovery rate is the uterine re-flushing procedure. To evaluate the effect of re-flushing and the influence of breed and operator on bovine embryo recovery rate, 38 Nelore and 19 Jersey females were stimulated using FSH, with embryos being collected by one of two trained operators. At the end of flushing, the catheter was sealed and maintained into the uterine body, filled with flushing medium, while females were released to a paddock for 30 to 50 min, to be submitted to the re-flushing procedure by the same operator. A total of 599 structures were recovered out of 57 procedures, from which 423 (70.6%) were obtained in the first flushing and 176 (29.4%) after re-flushing. Mean recovery rates of 7.4 and 3.1 structures were obtained after the first and second flushing, respectively, for a total of 10.5 structures per cow. Structures were obtained in 73.6% ( 42 out of 57) of the re-flushing procedures. No breed effect was observed on total ova or embryo recovery, with 10.9 total ova collected from Jersey and 10.3 from Nelore females. Likewise, the embryo recovery rate obtained following uterine flushing or re-flushing did not differ either between Jersey (8.1 and 2.7) and Nelore (7.0 and 3.2) animals, or between operators A (7.5 and 3.3) and B (7.3 and 2.9), respectively. In conclusion, the uterine re-flushing procedure significantly increased the rate of embryo recovery in Jersey and Nelore females, with no operator influence being observed.

Developmental Potential of Bovine Hand-Made Clone Embryos Reconstructed by Aggregation or Fusion with Distinct Cytoplasmic Volumes

Animal cloning has been associated with developmental abnormalities, with the level of heteroplasmy caused by the procedure being one of its potential limiting factors. The aim of this study was to determine the effect of the fusion of hemicytoplasts or aggregation of hemiembryos, varying the final cytoplasmic volume, on development and cell density of embryos produced by hand-made cloning (HMC), parthenogenesis or by in vitro fertilization (IVF). One or two enucleated hemicytoplasts were paired and fused with one skin somatic cell. Activated clone and zona-free parthenote embryos and hemiembryos were in vitro cultured in the well-of-the-well (WOW) system, being allocated to one of six experimental groups, on a per WOW basis: single clone or parthenote hemiembryos (1 x 50%); aggregation of two (2 x 50%), three (3 x 50%), or four (4 x 50%) clone or parthenote hemiembryos; single clone or parthenote embryos (1 x 100%); or aggregation of two clone or parthenote embryos (2 x 100%). Control zona-intact parthenote or IVF embryos were in vitro cultured in four-well dishes. Results indicated that the increase in the number of aggregated structures within each WOW was followed by a linear increase in cleavage, blastocyst rate, and cell density. The increase in cytoplasmic volume, either by fusion or by aggregation, had a positive effect on embryo development, supporting the establishment of pregnancies and the birth of a viable clone calf after transfer to recipients. However, embryo aggregation did not improve development on a hemicytoplast basis, except for the aggregation of two clone embryos.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

In vitro development of cloned bovine embryos produced by handmade cloning using somatic cells from distinct levels of cell culture confluence

The relationship between the level of cell confluence near the plateau phase of growth and blastocyst yield following somatic cell cloning is not well understood. We examined the effect of distinct cell culture confluence levels on in vitro development of cloned bovine embryos. In vitro-matured bovine oocytes were manually bisected and selected by DNA staining. One or two enucleated hemi-cytoplasts were paired and fused with an adult skin somatic cell. Cultured skin cells from an adult Nellore cow harvested at three distinct culture confluence levels (70-80, 80-90, and > 95%) were used for construction of embryos and hemi-embryos. After activation, structures were cultured in vitro as one embryo (1 x 100%) or as aggregates of two hemi-embryos (2 x 50%) per microwell. Fusion, cleavage and blastocyst rates were compared using the chi(2) test. The fusion rate for hemi-embryos (51.4%) was lower than for embryos (67.6%), with no influence of degree of cell confluence. However, blastocyst rates improved linearly (7.0, 17.5, and 29.4%) with increases in cell confluence. We conclude that degree of cell culture confluence significantly influences subsequent embryo development; use of a cell population in high confluence (> 90%) for nuclear transfer significantly improved blastocyst yield after cloning.

Serum-Starved Apoptotic Fibroblasts Reduce Blastocyst Production but Enable Development to Term after SCNT in Cattle

Cell cycle synchronization by serum starvation (SS) induces apoptosis in somatic cells. This side effect of SS is hypothesized to negatively affect the outcome of somatic cell nuclear transfer (SCNT). We determined whether apoptotic fibroblasts affect SCNT yields. Serum-starved, adult, bovine fibroblasts were stained with annexin V-FITC/propidium iodide to allow apoptosis detection by flow cytometry. Positive and negative cells sorted by fluorescence activated cell sorting (FACS) and an unsorted control group were used as nuclear donors for SCNT. Reconstructed embryos were cultured in vitro and transferred to synchronized recipients. Apoptosis had no effect on fusion and cleavage rates; however, it resulted in reductions in blastocyst production and quality measured by apoptotic index. However, reconstructed embryos with apoptotic cells resulted in pregnancy rates similar to that of the control on day 30, and generated one live female calf. In conclusion, we showed that apoptotic cells present in serum-starved cultures negatively affect embryo production after SCNT without compromising full-term development. Further studies will evaluate the ability of the oocyte to reprogram cells in specific phases of apoptosis.

Random X Inactivation and Extensive Mosaicism in Human Placenta Revealed by Analysis of Allele-Specific Gene Expression along the X Chromosome

Imprinted inactivation of the paternal X chromosome in marsupials is the primordial mechanism of dosage compensation for X-linked genes between females and males in Therians. In Eutherian mammals, X chromosome inactivation (XCI) evolved into a random process in cells from the embryo proper, where either the maternal or paternal X can be inactivated. However, species like mouse and bovine maintained imprinted XCI exclusively in extraembryonic tissues. The existence of imprinted XCI in humans remains controversial, with studies based on the analyses of only one or two X-linked genes in different extraembryonic tissues. Here we readdress this issue in human term placenta by performing a robust analysis of allele-specific expression of 22 X-linked genes, including XIST, using 27 SNPs in transcribed regions. We show that XCI is random in human placenta, and that this organ is arranged in relatively large patches of cells with either maternal or paternal inactive X. In addition, this analysis indicated heterogeneous maintenance of gene silencing along the inactive X, which combined with the extensive mosaicism found in placenta, can explain the lack of agreement among previous studies. Our results illustrate the differences of XCI mechanism between humans and mice, and highlight the importance of addressing the issue of imprinted XCI in other species in order to understand the evolution of dosage compensation in placental mammals.

Maternal diabetes affects cell proliferation in developing rat placenta

Placentation starts with the formation of a spheroidal trophoblastic shell surrounding the embryo, thus facilitating both implantation into the uterine stroma and contact with maternal blood. Although it is known that diabetes increases the placental size and weight, the mechanisms responsible for this alteration are still poorly understood. In mammals, cellular proliferation occurs in parallel to placental development and it is possible that diabetes induces abnormal uncontrolled cell proliferation in the placenta similar to that seen in other organs (e.g. retina). To test this hypothesis, the objective of this work was to determine cell proliferation in different regions of the placenta during its development in a diabetic rat model. Accordingly, diabetes was induced on day 2 of pregnancy in Wistar rats by a single injection of alloxan (40 mg/kg i.v.). Placentas were collected on days 14, 17, and 20 postcoitum. Immunoperoxidase was used to identify Ki67 nuclear antigen in placental sections. The number of proliferating cells was determined in the total placental area as well as in the labyrinth, spongiotrophoblast and giant trophoblast cell regions. During the course of pregnancy, the number of Ki67 positive cells decreased in both control and diabetic rat placentas. However, starting from day 17 of pregnancy, the number of Ki67 positive cells in the labyrinth and spongiotrophoblast regions was higher in diabetic rat placentas as compared to control. The present results demonstrate that placentas from the diabetic rat model have a significantly higher number of proliferating cells in specific regions of the placenta and at defined developmental stages. It is possible that this increased cell proliferation promotes thickness of the placental barrier consequently affecting the normal maternal-fetal exchanges.

Spatiotemporal patterns of macrophage migration inhibitory factor (Mif) expression in the mouse placenta

Background: Macrophage migration inhibitory factor (MIF) has special pro-inflammatory roles, affecting the functions of macrophages and lymphocytes and counter-regulating the effects of glucocorticoids on the immune response. The conspicuous expression of MIF during human implantation and early embryonic development also suggests this factor acts in reproductive functions. The overall goal of this study was to evaluate Mif expression by trophoblast and embryo placental cells during mouse pregnancy. Methods: Mif was immunolocalized at implantation sites on gestation days (gd) 7.5, 10.5, 13.5 and 17.5. Ectoplacental cones and fetal placentas dissected from the maternal tissues were used for Western blotting and qRT-PCR assays on the same gestation days. Results: During the post-implantation period (gd7.5), trophoblast giant cells showed strong Mif reactivity. In later placentation phases (gds 10.5-17.5), Mif appeared to be concentrated in the junctional zone and trophoblast giant cells. Mif protein expression increased significantly from gd7.5 to 10.5 (p = 0.005) and from gd7.5 to 13.5 (p = 0.03), remaining at high concentration as gestation proceeded. Higher mRNA expression was found on gd10.5 and was significantly different from gd13.5 (p = 0.048) and 17.5 (p = 0.009). Conclusions: The up-regulation of Mif on gd10.5 coincides with the stage in which the placenta assumes its three-layered organization (giant cells, spongiotrophoblast and labyrinth zones), fetal blood circulation begins and population of uNK cells reaches high proportions at the maternal counter part of the placenta, suggesting that Mif may play a role in either the placentation or in the adaptation of the differentiated placenta to the uterus or still in gestational immunomodulatory responses. Moreover, it reinforces the possibility of specific activities for Mif at the maternal fetal interface.

Design and fabrication of microcavity-array superhydrophobic surfaces

We have modeled, fabricated, and characterized superhydrophobic surfaces with a morphology formed of periodic microstructures which are cavities. This surface morphology is the inverse of that generally reported in the literature when the surface is formed of pillars or protrusions, and has the advantage that when immersed in water the confined air inside the cavities tends to expel the invading water. This differs from the case of a surface morphology formed of pillars or protrusions, for which water can penetrate irreversibly among the microstructures, necessitating complete drying of the surface in order to again recover its superhydrophobic character. We have developed a theoretical model that allows calculation of the microcavity dimensions needed to obtain superhydrophobic surfaces composed of patterns of such microcavities, and that provides estimates of the advancing and receding contact angle as a function of microcavity parameters. The model predicts that the cavity aspect ratio (depth-to-diameter ratio) can be much less than unity, indicating that the microcavities do not need to be deep in order to obtain a surface with enhanced superhydrophobic character. Specific microcavity patterns have been fabricated in polydimethylsiloxane and characterized by scanning electron microscopy, atomic force microscopy, and contact angle measurements. The measured advancing and receding contact angles are in good agreement with the predictions of the model. (C) 2010 American Institute of Physics. [doi:10.1063/1.3466979]