Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Microbial Culture in Minimal Medium With Oil Favors Enrichment of Biosurfactant Producing Genes.

The waste produced by petrochemical industries has a significant environmental impact. Biotechnological approaches offer promising alternatives for waste treatment in a sustainable and environment-friendly manner. Microbial consortia potentially clean up the wastes through degradation of hydrocarbons using biosurfactants as adjuvants. In this work, microbial consortia were obtained from a production water (PW) sample from a Brazilian oil reservoir using enrichment and selection approaches in the presence of oil as carbon source. A consortium was obtained using Bushnell-Haas (BH) mineral medium with petroleum. In parallel, another consortium was obtained in yeast extract peptone dextrose (YPD)-rich medium and was subsequently compared to the BH mineral medium with petroleum. Metagenomic sequencing of these microbial communities showed that the BH consortium was less diverse and predominantly composed of Brevibacillus genus members, while the YPD consortium was taxonomically more diverse. Functional annotation revealed that the BH consortium was enriched with genes involved in biosurfactant synthesis, while the YPD consortium presented higher abundance of hydrocarbon degradation genes. The comparison of these two consortia against consortia available in public databases confirmed the enrichment of biosurfactant genes in the BH consortium. Functional assays showed that the BH consortium exhibits high cellular hydrophobicity and formation of stable emulsions, suggesting that oil uptake by microorganisms might be favored by biosurfactants. In contrast, the YPD consortium was more efficient than the BH consortium in reducing interfacial tension. Despite the genetic differences between the consortia, analysis by a gas chromatography-flame ionization detector showed few significant differences regarding the hydrocarbon degradation rates. Specifically, the YPD consortium presented higher degradation rates of C12 to C14 alkanes, while the BH consortium showed a significant increase in the degradation of some polycyclic aromatic hydrocarbons (PAHs). These data suggest that the enrichment of biosurfactant genes in the BH consortium could promote efficient hydrocarbon degradation, despite its lower taxonomical diversity compared to the consortium enriched in YPD medium. Together, these results showed that cultivation in a minimal medium supplemented with oil was an efficient strategy in selecting biosurfactant-producing microorganisms and highlighted the biotechnological potential of these bacterial consortia in waste treatment and bioremediation of impacted areas.

Comparison between the additive effects of diluted (rFSH) and diluted/dynamized (FSH 6 cH) recombinant follicle-stimulating hormone on the in vitro culture of ovine preantral follicles enclosed in ovarian tissue.

OBJECTIVE: This study compared 2 types of recombinant follicle stimulating hormone (rFSH): diluted and diluted/dynamized, on in vitro development of ovine follicles. METHODS: In experiment 1, ovarian fragments were cultured for 1 or 7 days in α-MEM(+) in the absence or presence of different concentrations of diluted rFSH to determine the best concentration. In experiment 2, the effect of diluted and diluted/dynamized rFSH (rFSH 6 cH--ultradiluted and succussioned), alone or in combination, was studied. RESULTS: In experiment 1, compared to control, 50ng/mL of diluted rFSH induced higher rates of follicular survival after 7 days of culture and higher percentages of growing follicles at day 1 of culture (P<0.05). In experiment 2, compared to control, diluted/dynamized rFSH induced higher follicular diameter and survival rate after 7 days and early follicle activation at day 1 of culture (P<0.05). Compared to diluted rFSH, diluted/dynamized rFSH induced higher rates of follicle activation at day 1 of culture (P<0.05). CONCLUSION: In conclusion, compared to the control medium, diluted/dynamized rFSH promoted survival and early activation of follicles, while diluted rFSH promoted higher activation later in the culture. Thus, diluted/dynamized rFSH may be used as an alternative to diluted rFSH for the in vitro culture of ovine preantral follicles.

Análise fitoquímica e atividade antifúngica do óleo essencial de folhas de Lippia sidoides Cham. e do Timol contra cepas de Candida spp / Phytochemical analysis and antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains

RESUMO A resistência de fungos do gênero Candida aos fármacos químicos tem lançado o desafio de se identificar novas substâncias que possuam atividade antibiótica ou venham a modular o efeito de produtos atualmente usados contra candidíase. O presente estudo avaliou a atividade antifúngica do óleo essencial de Lippia sidoides Cham. e do timol, sobre cepas de Candida. Inicialmente os produtos foram testados frente a 16 cepas fúngicas pela técnica de difusão em meio sólido, o que permitiu selecionar linhagens para continuidade da pesquisa. Com as linhagens de Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) e Candida tropicalis (CT LM 20), procedeu-se, por microdiluição em caldo, a determinação da Concentração Inibitória Mínima (CIM) e em meio sólido, a Concentração Fungicida Mínima (CFM) dos produtos foi identificada. O microcultivo das leveduras em meio empobrecido foi realizado para verificação de alterações morfológicas e, além disso, uma análise da composição química do óleo foi realizada por Cromatografia Gasosa acoplada à espectrometria de massas (CG-EM). Nesta análise, o constituinte majoritário foi o timol (84,95%), seguido de compostos como p-cimeno e Éter metil carvacrol, entre outros. A CIM do óleo essencial de Lippia sidoides Cham. frente às cepas variou entre 64 e 256 μg/mL, enquanto a CIM do timol foi estabelecida entre 32 e 64 μg/mL. A CFM do óleo essencial foi determinada entre 128 e 512 μg/mL e para o timol foram encontrados valores entre 64 e 128 μg/mL. Em relação à análise micromorfológica, verificada nas concentrações de CIM e CIM x 2, o óleo essencial inibiu o dimorfismo das cepas CK 01 e CT 20 na CIM e quando foi ensaiado o timol, este, na CIM, impediu a transição morfológica das cepas CK 01 e CA 62. Uma redução da morfogênese também foi obsevada na cepa CT 20, porém apenas em CIM x 2 e de forma mais discreta. Os resultados enaltecem o potencial antifúngico de L. sidoides e de seu composto majoritário timol tanto no combate à Candida quanto na neutralização de um dos fatores de virulência, a capacidade invasiva por formação de hifas e pseudohifas verificado na condição patogênica da candidíase. Estes dados são promissores e poderão incentivar futuras pesquisas sobre os aspectos fitoquímicos, toxicológicos e farmacológicos tanto do óleo essencial de Lippia sidoides como também de seus componentes químicos.

ABSTRACT The resistance of the Candida against drugs has been a challenge to the discovery of new substances with antimicrobial or modulatory effects that could be used against the cadidiasis. This work evaluated the antifungal activity of the essential oil of Lippia sidoides Cham. and of the Thymol against Candida strains. The products were tested towards 16 strains of Candida using the diffusion method, which allowed to select the strains in order to proceed with the research. The strains of Candida krusei (CK LMBM 01, CK LMBM 02), Candida albicans (CA LM 62) and Candida tropicalis (CT LM 20) were assayed by the microdilution method so that the Minimal Inhibitory Concentration (MIC) and the Minimal Fungicide Concentration (MFC) could be determined. The morphogenesis of the Candida was evaluated using poor environment in order to observe morphological changes. The composition of the essential oil was determined by GC-MS. The main compound observed was the thymol (84.95%). The MIC of the essential oil of L. sidoides and Thymol ranged between 64 to 256 μg/mL, and between 32 to 64 μg/mL respectively. The MFC of the essential oil and the thymol varied between 128 to 512 μg/mL and 64 to 128 μg/mL respectively. The morphogenesis of different Candida strains was inhibited in the MIC and MICx2 to the essential oil and thymol. The results indicated the antifungal potential of the L. sidoides and of the Thymol due to the inhibition of the invasive capacity, one of the most important virulence factors for the candidiasis` development. These results are promising to new researches about the phytochemical, toxicological and pharmacological aspects of the essential oil of L. sidoides and its phytochemical compounds.

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles.

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM(+)) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM(+) alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM(+) alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.

Genotoxicity assessment in aquatic environment impacted by the presence of heavy metals.

The aim of this study was to access the genotoxic potential of Extremoz Lake waters in Northeastern Brazilian coast, using the Allium cepa system, piscine micronucleus test and comet assay. In addition, heavy metal levels were quantified by atomic absorption flame spectrometry. The results of the A. cepa system showed significant changes in the frequency of chromosome aberrations and in the mitotic index compared to negative control. No significant changes were observed in micronuclei frequency in the erythrocytes of Oreochromis niloticus. The comet assay showed a statistically significant alteration in the level of DNA breaks of O. niloticus. Chemical analysis detected an increase in heavy metal levels in different sampling periods. These results point out a state of deterioration of water quality at Extremoz Lake, caused by heavy metal contamination and genotoxic activity. It is recommended to establish a monitoring program for the presence of genotoxic agents in this water lake.