Specific Phylotypes of Saprolegnia parasitica Associated with Atlantic Salmon Freshwater Aquaculture.

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 oomycete and fungal isolates that were successfully cultured and sequenced from 14 aquaculture sites in Scotland across a two-year sampling period. An ITS phylogenetic analysis of all isolates was performed according to whether they were isolated from fish or water samples and during enzootic or epizootic periods. Several genera of oomycetes were isolated from sampling sites, including Achlya, Leptolegnia, Phytophthora, and Pythium, but by far the most prevalent was Saprolegnia, accounting for 66% of all oomycetes isolated. An analysis of the ITS region of Saprolegnia parasitica showed five distinct phylotypes (S2-S6); S1 was not isolated from any site. Phylotype S2 was the most common and most widely distributed phylotype, being found at 12 of the 14 sampling sites. S2 was overwhelmingly sampled from fish (93.5%) and made up 91.1% of all S. parasitica phylotypes sampled during epizootics, as well as 67.2% of all Saprolegnia. This study indicates that a single phylotype may be responsible for Saprolegnia outbreaks in Atlantic salmon fish farms, and that water sampling and spore counts alone may be insufficient to predict Saprolegnia outbreaks in freshwater aquaculture.

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration.

The regenerative therapies with stem cells (SC) has been increased by the cryopreservation, permitting cell storage for extended periods. However, the permeating cryoprotectant agents (CPAs) such as dimethylsulfoxide (DMSO) can cause severe adverse effects. Therefore, this study evaluated equine mesenchymal stem cells derived from adipose tissue (eAT-MSCs) in fresh (Control) or after slow freezing (SF) in different freezing solutions (FS). The FS comprise DMSO and non-permeating CPAs [Trehalose (T) and the SuperCool X-1000 (X)] in association or not, totalizing seven different FS: (DMSO; T; X; DMSO+T; DMSO+X; T+X, and DMSO+T+X). Before and after cryopreservation were evaluated, viability, colony forming unit (CFU), and cellular differentiation capacity. After freezing-thawing, the viability of the eAT-MSCs reduced (P< 0.05) in all treatments compared to the control. However, the viability of frozen eAT-MSCs in DMSO (80.3 ± 0.6) was superior (P<0.05) to the other FS. Regarding CFU, no difference (P>0.05) was observed between fresh and frozen cells. After freezing-thawing, the eAT-MSCs showed osteogenic, chondrogenic, and adipogenic lineages differentiation potential. Nonetheless, despite the significative reduction in the osteogenic differentiation capacity between fresh and frozen cells, no differences (P > 0.05) were observed among FS. Furthermore, the number of chondrogenic differentiation cells frozen in DMSO+X solution reduced (P<0.05) comparing to the control, without differ (P>0.05) to the other FS. The adipogenic differentiation did not differ (P>0.05) among treatments. In conclusion, although these findings confirm the success of DMSO to cryopreserve eAT-MSCs, the Super Cool X-1000 could be a promise to reduce the DMSO concentration in a FS.

As terapias regenerativas com células-tronco (CT) têm sido incrementadas pela criopreservação, permitindo o armazenamento celular. No entanto, os agentes crioprotetores (ACPs) penetrantes, como DMSO, podem causar efeitos adversos graves. Portanto, este estudo avaliou células-tronco mesenquimais equinas derivadas de tecido adiposo (CTM-TAe) in natura (Controle) ou após congelamento lento (CL) em diferentes soluções de congelamento (SC). As SCs compreendem DMSO e ACPs não permeáveis [Trealose (T) e o SuperCool X-1000 (X)] associados ou não: (DMSO; T; X; DMSO+T; DMSO+X; T +X e DMSO+T+X). Antes e após a criopreservação foram avaliados, viabilidade, unidade formadora de colônia (UFC) e capacidade de diferenciação celular. Após o congelamento-descongelamento, a viabilidade das CTM-TAe reduziu (P< 0,05) em todos os tratamentos em relação ao controle. Entretanto, a viabilidade das CTM-TAe congeladas em DMSO (80,3 ± 0,6) foi superior (P<0,05) às demais SC. Em relação às UFC, não houve diferença (P>0,05) entre células frescas e congeladas. Após congelamento-descongelamento, as CTM-TAe apresentaram potencial de diferenciação de linhagens osteogênicas, condrogênicas e adipogênicas. No entanto, apesar da redução significativa na capacidade de diferenciação osteogênica entre células frescas e congeladas, não foram observadas diferenças (P > 0,05) entre SCs. Além disso, o número de células de diferenciação condrogênica congeladas em solução de DMSO+X reduziu (P<0,05) em relação ao controle, sem diferir (P>0,05) das demais SCs. A diferenciação adipogênica não diferiu (P>0,05) entre os tratamentos. Em conclusão, embora esses achados confirmem o sucesso do DMSO para criopreservar CTM-TAe, o Super Cool X-1000 pode ser uma promessa para reduzir a concentração de DMSO.

Morphology and immunolocalization of intertubular steroidogenic cell in mesonephros of Podocnemis expansa during gonadal differentiation.

Sex steroid hormones are critical in gonadal differentiation in turtles. The gonads are not the only organs responsible for producing these hormones during this phase. Mesonephros play an important role in steroidogenesis. The present study aimed to investigate the presence of steroidogenic cells in mesonephros of Podocnemis expansa during gonadal differentiation and to evaluate their morphology and ultrastructure. Ten embryos of P. expansa were collected from 5 nests on day 36 of incubation, during spawning period on an artificial beach. Embryos were extracted from eggs by slicing the shell and euthanized. They were dissected under a stereoscopic microscope to collect the gonad-mesonephro complex, in which were fixed and subsequently processed for light microscopy, immunohistochemistry and transmission electron microscopy analysis. During histological analysis was observed mesonephros has typical morphological structure. Immunohistochemistry showed immunoreaction to aromatase in cells of intertubular space. Confirming these findings, it was possible to observe a type of intertubular cell in several regions of mesonephro, being more predominant in region close to blood vessels, distal and proximal tubules. In ultrastructural analysis these cells were characterized by having a clear, large, and rounded nucleus with evident nucleolus and cytoplasm rich in electron-dense droplets. This study demonstrated for the first time the presence of cells with morphological, immunohistochemical and ultrastructural characteristics similar to steroid-producing cells in P. expansa mesonephrons, suggesting that this organ may contribute to gonadal differentiation in this species.

Evaluation of Potential Transfer of the Pathogen Saprolegnia parasitica between Farmed Salmonids and Wild Fish.

Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms.

Development of a 3D spheroid cell culture system from fish cell lines for in vitro infection studies: Evaluation with Saprolegnia parasitica.

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens.

Saprolegnia infection after vaccination in Atlantic salmon is associated with differential expression of stress and immune genes in the host.

This study assessed the impact of routine vaccination of Atlantic salmon pre-smolts on gene expression and the possible link to saprolegniosis on Scottish fish farms. Fish were in 4 different groups 1) 'control' - fish without handling or vaccination 2) 'vaccinated' - fish undergoing full vaccination procedure 3) 'non vaccinated' - fish undergoing full vaccination procedure but not vaccinated and 4) 'vaccinated-MH' - fish undergoing vaccination, but procedure involved minimal handling. A strong increase in cortisol and glucose levels was observed after 1 h in all groups relative to the control group. Only in the non-vaccinated group did the level decrease to near control levels by 4 h. Expression levels of six stress marker genes in general for all groups showed down regulation over a 9-day sampling period. In contrast, expression levels for immune response genes in the head kidney showed significant up-regulation for all eight genes tested for both vaccinated groups whereas the non-vaccinated group showed up-regulation for only MHC-II and IL-6b in comparison to the control. Both the vaccination procedure and the administration of the vaccine itself were factors mediating changes in gene expression consistent with fish being susceptible to natural occurring saprolegniosis following vaccination.

BMP-15 activity on in vitro development of collared peccary (Pecari tajacu Linnaeus, 1758) preantral follicles.

This study investigated the effects of BMP-15 on the in vitro development of preantral follicles of collared peccaries. Ovarian fragments were cultured for 1 or 6 days in Tissue Culture Medium 199 (TCM199+ ) supplemented with BMP-15 at rates of 0, 1, 25 or 50 ng/ml. The fragments were analysed histologically by evaluating follicular morphology, activation and growth as well as the potential for proliferation of granulosa cells. Our results show the addition of 25 ng/ml BMP-15 in the medium provided the greatest percentage of normal follicles (79.67% ± 0.69) when compared to other treatments (p < .05); however, this result is similar to 1 ng/ml BMP-15 (74.00% ± 1.90, p > .05). Moreover, 25 and 50 ng/ml of BMP-15 promoted follicular activation. BMP-15 supplements did not affect oocyte and follicular growth. All concentrations of BMP-15 increased the number of nucleolus organizer regions (NORs) after 1 day of culture when compared to fresh fragments or the control samples (p < .05). However, at the end of the experiment, the number of NORs in follicles cultured in all treatments was higher than that observed in the fresh control (sample taken prior to culturing) (p > .05). In summary, the addition of 25 ng/ml BMP-15 to the culture medium of collared peccary preantral follicles maintained a high number of morphologically healthy follicles and stimulated the activation of primordial follicles after 6 days in culture.

Establishment of a protocol for the isolation of ovarian preantral follicles derived from collared peccaries (Pecari tajacu).

We compare the efficiency of mechanical or enzymatic methods, and their combination, for the isolation of ovarian preantral follicles (PFs) from collared peccaries. The ovaries from six females were subjected to the different methods investigated here. For the enzymatic method, ovary fragments were exposed to collagenase type IV in TCM-HEPES medium; the mechanical procedure was based on ovarian cortex dissociation by using a scalpel blade. The residual solution obtained after the mechanical isolation was subjected to the enzymatic procedure. The number of isolated PFs was quantified and classified as primordial, primary, or secondary; their viability was assessed using trypan blue dye assay. To confirm the results, PFs derived from the most efficient method were evaluated for integrity using scanning electron microscopy (SEM) and subjected to a 24 h in vitro culture for subsequent evaluation of viability by using fluorescent probes. A higher number of PFs (P < 0.05) was obtained from the enzymatic method (961.7 ± 132.9) in comparison with the mechanical method (434.3 ± 88.9), but no difference was observed between the two methods and their combination (743.2 ± 92.8). The trypan blue assay showed that the enzymatic method (98.7 ± 0.6%) provided the highest percentage of viable follicles (P < 0.05). Furthermore, SEM confirmed the ultrastructural integrity of the surface architecture of peccary PFs isolated by the enzymatic procedure; epifluorescence microscopy was used to confirm their viability (86.0%). In conclusion, we suggest that the enzymatic method investigated here is useful for the isolation of viable ovarian PFs from collared peccaries.

Advances and Challenges of Using Ovarian Preantral Follicles to Develop Biobanks of Wild Mammals.

The extinction rate of mammalian species has been accelerated in recent decades. It therefore is important to preserve and store genetic materials in cryobanks for research purposes and subsequent production of offspring through assisted reproductive techniques. Along with the systematic collection and storage of germplasm, research efforts focusing on in vitro culture to produce mature gametes are critical. Specifically, obtaining mature oocytes from in vitro culture of preantral follicles is a great option to enhance female fertility preservation since these early follicles represent 90%-95% of the follicular population on ovarian cortex biopsy. This review presents current advances and discusses limitations and prospects about isolation, cryopreservation/banking, and in vitro culture of preantral follicles from wild species.

Exophiala angulospora infection in hatchery-reared lumpfish (Cyclopterus lumpus) broodstock.

Samples from moribund lumpfish were collected in a marine hatchery in Scotland in 2015. Black nodules were noted on the skin, and gills and fungal hyphae were extensively distributed in musculature and internal organs. Multifocal chronic inflammatory lesions displaced structures in all affected organs. Mortalities commenced on completion of spawning in May and were evenly distributed over the second year in the temperature range 11-15°C. The main systemic infection causing agent was initially identified based on morphological characteristics as an Exophiala species. Ribosomal DNA (rDNA) ITS regions of the isolates were subsequently sequenced confirming the isolates belonged to Exophiala genus. All isolates fell in a single phylogenetic cluster, which is represented by Exophiala angulospora. Fish were treated with either formalin or Bronopol or a combination of both, but there was no effect on the pattern or numbers of mortalities. Isolates were also tested against three different concentrations of Latrunculin A, Amphotericin B and Itraconazole with no success. It is of utmost importance to increase the knowledge on pathogen-host interactions to successfully develop sustainable control methods.

Vitrification of ovarian tissue of Brazilian North-eastern donkeys (Equus asinus) using different cryoprotectants.

The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p < 0.05). When comparing treatments, the use of DMSO 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (72.3 ± 18.0%; p < 0.05). When vitrified using the DMSO + EG combination, a higher percentage (62.5 ± 29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p < 0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3 M + EG 3 M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.

Expression of markers for germ cells and oocytes in cow dermal fibroblast treated with 5-azacytidine and cultured in differentiation medium containing BMP2, BMP4 or follicular fluid.

This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 µM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 µM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 µM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.

Effects of bone morphogenetic protein 4 (BMP4) on in vitro development and survival of bovine preantral follicles enclosed in fragments ovarian tissue.

The aim of this study was to evaluate the effects of different concentrations of BMP4 on activation, development and mRNA expression of GDF9, BMP15, PCNA, Bax and Bcl2 in cultured bovine follicles enclosed in ovarian tissues. Ovarian tissue fragments were cultured for 6 days in α-MEM+ alone or supplemented with different concentrations of BMP4 (10, 50 or 100 ng/ml). Classical histology was performed to analyze follicle growth and morphology, while real-time PCR was used to analyze mRNA levels in fresh and cultured tissues. After 6 days, the culture of ovarian tissue in α-MEM+ alone or supplemented with 10, 50 or 100 ng/ml BMP4 promoted follicular activation. The different concentrations of BMP4 maintained the percentage of normal follicles similar to results of the control. The presence of 100 ng/ml BMP-4 in culture medium increased oocyte and follicular diameters of primary and secondary follicles when compared with those follicles from uncultured control or cultured in α-MEM+ alone (P < 0.05). The tissues cultured in the presence of increasing concentrations of BMP4 had an increase in mRNA expression of the tested genes, but despite this the differences were not statistically significant. In conclusion, 100 ng/ml BMP4 promotes an increase in diameters of follicles and oocytes of primary and secondary follicles after 6 days of in vitro culture.

Bailout intravenous esmolol for heart rate control in cardiac computed tomography angiography.

OBJECTIVE: To evaluate the efficacy and safety of a heart rate (HR) reduction protocol using intravenous esmolol as bailout for failed oral metoprolol regimens in patients undergoing coronary computed tomography angiography (CCTA) with 64-slice multidetector computed tomography (64-MDCT). METHODS: Patients who underwent cardiac 64-MDCT in a single institution between 2011 and 2014 were analyzed. Those with HR above 60 beats per minute (bpm) on presentation received oral metoprolol (50-200 mg) at least one hour before CCTA. Intravenous esmolol 1-2 mg/kg was administered as a bolus whenever HR remained over 65 bpm just before imaging. The primary efficacy endpoint was HR <65 bpm during CCTA. The primary safety endpoint was symptomatic hypotension or bradycardia up to hospital discharge. RESULTS: During the study period CCTA was performed in 947 cases. In 86% of these, oral metoprolol was the only medication required to successfully reduce HR <60 bpm. Esmolol was used in the remaining 130 patients (14%). For esmolol-treated patients mean baseline and acquisition HR were 74±14 bpm and 63±9 bpm, respectively (p<0.001). The target HR of <65 bpm was achieved in 82 of the 130 esmolol-treated patients (63%). Considering the whole population, esmolol use led to a significant increase in the primary efficacy endpoint from 86% to 95% (p<0.001). Esmolol also resulted in a statistically, but not clinically, significant reduction in systolic blood pressure (144±22 to 115±17 mmHg; p<0.001). The combined primary safety endpoint was only observed in two (1.5%) patients. CONCLUSION: Despite optimal use of oral beta-blockers, 14% of patients needed intravenous esmolol for HR control. The pre-medication combination of oral metoprolol and on-demand administration of intravenous esmolol was safe and effective and enabled 95% of patients to be imaged with HR below 65 bpm.

Influence of BMP-2 on early follicular development and mRNA expression of oocyte specific genes in bovine preantral follicles cultured in vitro.

This study evaluates the effect of different concentrations (0, 10, 50 and 100ng/mL) of bone morphogenetic protein-2 (BMP-2) on primordial and secondary follicle development. It also investigates the effects of FSH and BMP-2 on the growth, morphology, ultrastructure and expression of mRNA for GDF9, NLRP5 and NPM2 genes in secondary follicles cultured for 18 days. The presence of BMP-2 at all tested concentrations increased the development of primordial follicles in vitro, but the highest concentration of BMP-2 (100 ng/mL) reduced the percentage of normal follicles when compared with tissues cultured with 10 ng/mL BMP-2. During culture of secondary follicles, in contrast to higher concentrations (50 or 100 ng/mL), 10 ng/mL BMP-2 kept the morphology of follicles during initial stages of in vitro culture. This concentration of BMP-2 also benefits maintenance of the ultrastructure of 18-day cultured follicles. The presence of both BMP-2 and FSH in culture medium resulted in a significant (P<0.05) increase in follicular diameter after 18 days of culture. However, both FSH and BMP-2 reduced follicular mRNA expression of GDF9 and NLRP5 when compared to follicles cultured in media containing only FSH. In combination with FSH, BMP-2 reduced the mRNA levels of NPM2, when compared to follicles cultured in control medium. It is concluded from these data that 10 ng/mL BMP-2 promotes the growth of primordial in vitro and it helps to maintain the ultrastructure of secondary follicles, while FSH is more important for better expression of follicular markers like GDF9 and NLRP5.

The bone morphogenetic protein system and the regulation of ovarian follicle development in mammals.

The bone morphogenetic protein (BMP) family consists of several growth factor proteins that belong to the transforming growth factor-ß (TGF-ß) superfamily. BMPs bind to type I and type II serine-threonine kinase receptors, and transduce signals through the Smad signalling pathway. BMPs have been identified in mammalian ovaries, and functional studies have shown that they are involved in the regulation of oogenesis and folliculogenesis. This review summarizes the role of the BMP system during formation, growth and maturation of ovarian follicles in mammals.

Residência multiprofissional tecendo práticas interdisciplinares na prevenção da violência / Weaving interdisciplinarity practices into the avoidance of violence in the multi-professional residence

INTRODUÇÃO: A complexidade da atenção às situações de violência requer o investimento na integralidade das ações e do cuidado, lançando às equipes multiprofissionais o desafio da construção de uma prática interdisciplinar em conjunto com a escola. RELATO DE EXPERIÊNCIA: Analisar a integralidade da atenção à saúde e à educação a partir da ação das residentes do Programa da Residência Integrada Multiprofissional em Saúde da Criança com transversalidade em violências e vulnerabilidades inseridas numa equipe de Estratégia de Saúde da Família no Município de Porto Alegre e na escola estadual pertencente ao seu território. Trata-se da experiência interdisciplinar desenvolvida em dois níveis de abordagem: com os alunos do 1° ao 4° ano através de atividades lúdicas; e com os pais e responsáveis através da roda de conversa. A base da intervenção foram as oficinas com os alunos, buscando problematizar, através de intervenções lúdicas e brincadeiras, as situações de violência percebidas nos seus diversos ambientes. CONCLUSÃO: A prática interdisciplinar de forma consciente permite a organização do processo de trabalho com o escopo da prática colaborativa em projeto de educação interprofissional.

INTRODUCTION: The complexity of the attention to the situations of violence requires the investment in actions and care integrity, launching to the multi-professional teams the challenge of building an interdisciplinary practice together with the school. EXPERIENCE REPORT: To analyze the integrity of the attention to health and education of the residents' actions in the Program of the Multi-professional Integrated Residence in Children's Health, which are exposed to violence and vulnerability and inserted into a team of Family Health Strategy in Porto Alegre District, and the Public School that belongs to its area. It works on the inter-course studying experience developed inside two approaching levels: one with the primary level children through playing activities, and the other with their parents or guardians through conversations. The support for this intervention was the workshops with the students, aiming at bringing down the violent situations in their different environments through playing games. CONCLUSION: The interdisciplinary practice in a conscious way allows the organization of the work process with the scope of the collaborative practice in one inter-professional education project.

Activin-A promotes the development of goat isolated secondary follicles in vitro.

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 µm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

Effects of jacalin and follicle-stimulating hormone on in vitro goat primordial follicle activation, survival and gene expression.

This study aims to investigate the effects of jacalin and follicle-stimulating hormone (FSH) on activation and survival of goat primordial follicles, as well as on gene expression in cultured ovarian tissue. Ovarian fragments were cultured for 6 days in minimum essential medium (MEM) supplemented with jacalin (10, 25, 50 or 100 µg/ml - Experiment 1) or in MEM supplemented with jacalin (50 µg/ml), FSH (50 ng/ml) or both (Experiment 2). Non-cultured and cultured tissues were processed for histological and ultrastructural analysis. Cultured tissues from Experiment 2 were also stored to evaluate the expression of BMP-15, KL (Kit ligand), c-kit, GDF-9 and proliferating cell nuclear antigen (PCNA) by real-time polymerase chain reaction (PCR). The results of Experiment 1 showed that, compared with tissue that was cultured in control medium, the presence of 50 µg/ml of jacalin increased both the percentages of developing follicles and viability. In Experiment 2, after 6 days, higher percentages of normal follicles were observed in tissue cultured in presence of FSH, jacalin or both, but no synergistic interaction between FSH and jacalin was observed. These substances had no significant effect on the levels of mRNA for BMP-15 and KL, but FSH increased significantly the levels of mRNA for PCNA and c-kit. On the other hand, jacalin reduced the levels of mRNA for GDF-9. In conclusion, jacalin and FSH are able to improve primordial follicle activation and survival after 6 days of culture. Furthermore, presence of FSH increases the expression of mRNA for PCNA and c-kit, but jacalin resulted in lower GDF-9 mRNA expression.

N-acetylcysteine improves morphologic and functional aspects of ovarian grafts in rats.

PURPOSE: To evaluate morphological and functional aspects of the ovarian graft in transplanted rats treated with NAC. METHODS: Female Wistar rats, virgin, 3 to 4 months old, weighing 200-250 grams were used in experiments. The rats have been kept in proper sanitary conditions, receiving food and water ad libitum. Five groups (n=10, each) were constituted: 4 groups treated subcutaneously with NAC, at doses of 150, 300, 600 and 1200 mg/kg (NAC150, NAC300, NAC600 and NAC1200, respectively), one hour of before the ovarian transplantation and control group (GTx) - treated with physiological solution and submitted to ovarian transplantation. The rats were anesthetized and submitted to autologous left ovarian transplantation, without anastomosis in retroperitoneum, and contralateral oophorectomy. During follow-up of 4 or 15 days, the estrous cycle was evaluated by vaginal smears to determine cycle regularity. At the end of 4th or 15th days, rats were re-anesthetized and blood and graft were obtained to estradiol analysis and morphological assessment. Data were analysed by One Way Analysis of Variance (ANOVA) or ANOVA on ranks complemented by Student-Newman-Keuls test. RESULTS: At 4th day, viable follicles in the graft did not altered by NAC treatments. The NAC300 and NAC600 groups showed increasing in follicle atresia (p=0.012) compared to GTx and NAC1200 group. At 15th day, 50% of GTx, NAC150, and NAC300 rats showed regular oestrous cycle; 83% of NAC600 and 100% of NAC1200 rats returned to regular cycle. NAC1200 group showed increasing in primordial follicle compared to GTx, NAC150 or NAC300 (p=0.011). NAC did not interfere in estradiol levels after 4 or 15 days of transplantation. CONCLUSION: In autologous ovarian transplantation, high dose of NAC promotes graft viability with recovery of estrous cycle.