Saccharomyces cerevisiae as a probiotic agent and a possible aflatoxin B1 adsorbent in simulated fish intestinal tract conditions / Saccharomyces cerevisiae como agente probiótico e possível adsorvente de aflatoxina B1 em condições simuladas do trato intestinal de peixes

The aim of this study was to evaluate in vitro the probiotic potential and absorption of Saccharomyces cerevisiae for the aflatoxin B1 in simulated fish intestinal tract conditions. Three yeast strains were used, two from brewery: S. cerevisiae RC1 and S. cerevisiae RC3 and one from a fish farming environment: S. cerevisiae A8L2. The selected yeasts were subjected to the following in vitro tests: homologous inhibition, self-aggregation, co-aggregation, antibacterial activity, gastrointestinal conditions tolerance and adsorption of AFB1. All S. cerevisiae strains showed good capability of self-aggregation and co-aggregation with pathogenic bacteria. All yeast strains were able to survive the gastrointestinal conditions. In acidic conditions, the factors (strain vs. time) had interaction (P=0.0317), resulting in significant variation among the strains tested in the time periods analyzed. It was observed that there was also interaction (P=0.0062) in intestinal conditions, with an increased number of cells in the 12-hour period for all strains tested. In the adsorption test, the A8L2 strain was statistically more effective (P<0.005) for both AFB1 concentrations evaluated in this study (10 and 25ng/mL). Thus, it was observed that the strains of S. cerevisiae have potential probiotic and adsorbent of AFB1.(AU)

Objetivou-se, com esta pesquisa, avaliar in vitro o potencial probiótico e adsorvente de Saccharomyces cerevisiae para aflatoxina B1 em condições simuladas do trato intestinal de peixes. Foram utilizadas três cepas de leveduras, sendo duas provenientes de cervejaria: S. cerevisiae RC1 e S. cerevisiae RC3, e uma de ambiente de piscicultura: S. cerevisiae A8L2. As leveduras selecionadas foram submetidas aos seguintes testes in vitro: inibição homóloga, autoagregação, coagregação, atividade antibacteriana, viabilidade às condições gastrointestinais e adsorção de AFB1. Todas as estirpes de S. cerevisiae mostraram boa capacidade de autoagregação e coagregação com bactérias patogênicas. Todas as estirpes de levedura foram capazes de sobreviver às condições gastrointestinais. Em condições ácidas, os fatores (cepa x tempo) tiveram interação (P=0,0317), resultando em variações significativas entre as cepas testadas nos períodos de tempo analisados. Observou-se que também houve interação (P=0,0062) em condições intestinais, havendo um aumento do número de células no período de 12h para todas as cepas avaliadas. No ensaio de adsorção, a estirpe A8L2 foi a mais eficaz estatisticamente (P<0,005), para as duas concentrações de AFB1 avaliadas neste estudo (10 e 25ng. mL-1). Dessa forma, conclui-se que as cepas de Saccharomyces cerevisiae possuem potencial probiótico e adsorvente de AFB1.(AU)

Use of testicular tone and echogenicity in identifying boars with low sperm quality / Uso de tônus e ecogenicidade testiculares na identificação de reprodutores suínos com baixa qualidade espermática

This study aimed to evaluate the use of some testicular traits to identify boars with low sperm morphological quality. The consistency (scores from 1 to 5), tone with tonometry (mm), parenchyma echogenicity and heterogeneity with ultrasound (pixel) were assessed in 402 mature boars (18.5 months on average). Sperm abnormality thresholds (≤ 25% of total sperm abnormalities, ≤ 5% of heads, acrosome, neck or midpiece defects, ≤ 10% tail defects, and 15% cytoplasmic droplets) were used to classify boars as approved or disapproved. Three classes of testicular traits were formed (extremely low and high values, approximately 15% each, were kept in separated classes). When the traits were individually evaluated, fewer boars were approved if the echogenicity or heterogeneity were high, or if the tone was rigid. When evaluated in combination, the interaction between heterogeneity and tone, and between heterogeneity and echogenicity were significant. The high heterogeneity combined with soft tone or with hypo-echogenicity resulted in lower approval of boars. Tonometry and ultrasonography have a moderate potential to be included in breeding soundness examination of boars. When combined, they provide more reliable information about the impact of testicular parenchymal alterations on morphology of sperm cells.(AU)

O estudo objetivou avaliar o uso de características testiculares para identificar reprodutores com baixa qualidade de morfologia espermática. A consistência (escore de 1 a 5), o tônus por tonometria (mm), a ecogenicidade e a heterogeneidade do parênquima testicular por ultrassonografia (pixel) foram avaliados em 402 machos suínos maduros (18,5 meses, em média). Limiares máximos de anormalidades espermáticas (≤ 25% de defeitos totais, ≤ 5% de defeitos de cabeça, acrossoma, colo e peça intermediária, ≤ 10% de defeitos de cauda e 15% de gota citoplasmática) foram utilizados para classificar os machos como aprovados ou reprovados. Três classes de características testiculares foram formadas (valores extremamente baixos e altos, aproximadamente 15% em cada, foram mantidos em classes separadas). Quando as características testiculares foram avaliadas individualmente, menos machos foram aprovados se a ecogenicidade ou a heterogeneidade foram altas, ou se o tônus era rígido. Quando avaliadas em combinação, a interação entre heterogeneidade e tônus e a interação entre heterogeneidade e ecogenicidade foram significativas. A alta heterogeneidade combinada com testículos flácidos ou com testículos hipoecogênicos resultou em menor aprovação de cachaços. A tonometria e a ultrassonografia possuem potencial moderado para serem incluídas no exame andrológico de cachaços. Quando combinadas, as técnicas fornecem uma informação mais consistente do impacto das alterações do parênquima testicular na morfologia das células espermáticas.(AU)

Effect of Quercetin Rich Onion Extracts on Bacterial Quorum Sensing.

Quorum sensing (QS) regulates bacterial gene expression and studies suggest quercetin, a flavonol found in onion, as a QS inhibitor. There are no studies showing the anti-QS activity of plants containing quercetin in its native glycosylated forms. This study aimed to evaluate the antimicrobial and anti-QS potential of organic extracts of onion varieties and its representative phenolic compounds quercetin aglycone and quercetin 3-ß-D-glucoside in the QS model bacteria Chromobacterium violaceum ATCC 12472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MG1. Three phenolic extracts were obtained: red onion extract in methanol acidified with 2.5% acetic acid (RO-1), white onion extract in methanol (WO-1) and white onion extract in methanol ammonium (WO-2). Quercetin 4-O-glucoside and quercetin 3,4-O-diglucoside were identified as the predominant compounds in both onion varieties using HPLC-DAD and LC-ESI-MS/MS. However, quercetin aglycone, cyanidin 3-O-glucoside and quercetin glycoside were identified only in RO-1. The three extracts showed minimum inhibitory concentration (MIC) values equal to or above 125 µg/ml of dried extract. Violacein production was significantly reduced by RO-1 and quercetin aglycone, but not by quercetin 3-ß-D-glucoside. Motility in P. aeruginosa PAO1 was inhibited by RO-1, while WO-2 inhibited S. marcescens MG1 motility only in high concentration. Quercetin aglycone and quercetin 3-ß-D-glucoside were effective at inhibiting motility in P. aeruginosa PAO1 and S. marcescens MG1. Surprisingly, biofilm formation was not affected by any extracts or the quercetins tested at sub-MIC concentrations. In silico studies suggested a better interaction and placement of quercetin aglycone in the structures of the CviR protein of C. violaceum ATCC 12472 than the glycosylated compound which corroborates the better inhibitory effect of the former over violacein production. On the other hand, the two quercetins were well placed in the AHLs binding pockets of the LasR protein of P. aeruginosa PAO1. Overall onion extracts and quercetin presented antimicrobial activity, and interference on QS regulated production of violacein and swarming motility.

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle.

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well-established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6 h after systemic GnRH treatment, but decreased at 12 and 24 h to similar levels observed in samples collected at 0 h. After GnRH treatment, NPR1 mRNA was upregulated at 24 h, NPR3 mRNA gradually decreased after 3 h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24 h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.

Glyceroneogenesis in the hepatopancreas of the crab Neohelice granulata: Diet, starvation and season effects.

We determined the activity of glyceroneogenesis from [2-14C]-pyruvate, the phosphoenolpyruvate carboxykinase activity, [2-14C]-pyruvate oxidation and total lipid levels in the hepatopancreas of the crab Neohelice granulata fed with a carbohydrate-rich (HC) diet or a high-protein (HP) diet and then subjected to 5weeks of starvation, in summer and winter, to determine whether the seasonal adjustments of lipid metabolism to food scarcity are modulated by the composition of the diet previously given to the crabs. The results demonstrated that glyceroneogenesis is an active pathway in N. granulata hepatopancreas, and is regulated by seasonal variations, diet composition and starvation. This study showed that in summer the increase in the hepatopancreas glyceroneogenesis activity is among the strategies used by N. granulata fed an HP diet, to maintain the triglyceride/fatty acid cycle during starvation, a normal condition in the biological cycle of this crab. However, the administration of an HC diet reduced the glyceroneogenesis capacity in response to starvation in summer. In winter, the decrease in the glyceroneogenesis capacity in both fed (HP and HC diets) and starved crabs seems to be a strategy to reduce energy consumption and/or requirement. In contrast to the summer results, the incorporation of [2-14C]-pyruvate into 14CO2 was markedly higher in both diet (HC and HP) groups and in starved crabs during the winter. Four decades after the first study describing the glyceroneogenesis pathway in rat white adipose tissue, this pathway is evidenced for the first time in a crustacean.

mRNA expression profile of the TNF-α system in LH-induced bovine preovulatory follicles and effects of TNF-α on gene expression, ultrastructure and expansion of cumulus-oocyte complexes cultured in vitro.

This study evaluated (1) the effects of in vivo GnRH treatment on mRNA expression of TNF-α system (TNF-α, TNFR1 and TNFR2) in granulosa cells of bovine preovulatory follicles, (2) the in vitro influence of gonadotropins on mRNA expression of TNF-α system in cultured cumulus cells, (3) the protein expression of the TNF-α system in late antral follicles and, (4) the influence of TNF-α on cumulus cells expansion, ultrastructure and on expression of HAS2, CASP3 and CASP6 in follicular cells cultured for 24 h. An increased expression of TNF-α and TNFR1 was observed after 3, 6 and 12 h of GnRH treatment when compared to 0 and 24h. Higher TNFR2 mRNA levels were observed 3, 6 and 12 h after GnRH, when compared to 0 and 24 h. Proteins of TNF-α system were also expressed in late antral follicles. In vitro, TNF-α did not affect cumulus cells expansion, but reduced the HAS2, CASP3 and CASP6 mRNA levels in cumulus cells after 12 h. After 24 h of culture, TNF-α increased the mRNA levels for CASP6 in mural granulosa cells, while the TNF-α, TNFR1 and TNFR2 mRNA levels were increased in cumulus-oocyte complexes (COCs) cultured for 12 h with gonadotropins, but not after 24 h. Ultrastructural analysis confirmed the integrity of COCs cultured in presence of TNF-α. In conclusion, TNF-α system members are present in bovine antral follicles and expression of TNF-α is influenced by gonadotropins in vivo and in vitro. In vitro, TNF-α maintained cumulus cells ultrastructure during COC culture.

Mechanistic target of rapamycin is activated in bovine granulosa cells after LH surge but is not essential for ovulation.

The LH surge induces functional and morphological changes in granulosa cells. Mechanistic target of rapamycin (mTOR) is an integrator of signalling pathways in multiple cell types. We hypothesized that mTOR kinase activity integrates and modulates molecular pathways induced by LH in granulosa cells during the preovulatory period. Cows were ovariectomized and granulosa cells collected at 0, 3, 6, 12 and 24 hr after GnRH injection. While RHEB mRNA levels increased at 3 and 6 hr, returning to basal levels by 12 hr after GnRH treatment, RHOA mRNA levels increased at 6 hr and remained high thereafter. Western blot analyses revealed increased S6K phosphorylation at 3 and 6 hr after GnRH injection. Similarly, mRNA levels of ERK1/2, STAR and EGR-1 were higher 3 hr after GnRH treatment. Rapamycin treatment inhibited mTOR activity and increased AKT activity, but did not alter ERK1/2 phosphorylation and EGR1 protein levels in cultured bovine granulosa cells. Rapamycin also inhibited LH-induced increase in EREG mRNA abundance in granulosa cells in vitro. However, intrafollicular injection of rapamycin did not suppress ovulation. These findings suggest that mTOR is involved in the control of EREG expression in cattle, which may be triggered by LH surge stimulating RHEB and S6K activity.