MicroRNA qPCR normalization in Nile tilapia (Oreochromis niloticus): Effects of acute cold stress on potential reference targets.

The Nile tilapia (Oreochromis niloticus) is one of the most important cultured fish worldwide, but tilapia culture is largely affected by low temperatures. Recent studies suggest that microRNAs (miRNAs) regulate cold tolerance traits in fish. In general, qPCR-based methods are the simplest and most accurate forms of miRNA quantification. However, qPCR data heavily depends on appropriate normalization. Therefore, the aim of the present study is to determine whether the expression of previously tested, stably expressed miRNAs are affected by acute cold stress in Nile tilapia. For this purpose, one small nuclear RNA (U6) and six candidate reference miRNAs (miR-23a, miR-25-3, Let-7a, miR-103, miR-99-5, and miR-455) were evaluated in four tissues (blood, brain, liver, and gills) under two experimental conditions (acute cold stress and control) in O. niloticus. The stability of the expression of each candidate reference miRNA was analyzed by four independent methods (the delta Ct method, geNorm, NormFinder, and BestKeeper). Further, consensual comprehensive ranking of stability was built with RefFinder. Overall, miR-103 was the most stable reference miRNA in this study, and miR-103 and Let-7a were the best combination of reference targets. Equally important, Let-7a, miR-23a, and miR-25-3 remained consistently stable across different tissues and experimental groups. Considering all variables, U6, miR-99-5, and miR-455 were the least stable candidates under acute cold stress. Most important, suitable reference miRNAs were validated in O. niloticus, facilitating further accurate miRNA quantification in this species.

Chronic cold exposure modulates genes related to feeding and immune system in Nile tilapia (Oreochromis niloticus).

Nile tilapia is the fourth most produced species in the global aquiculture panorama. This species requires water temperatures higher than 16 °C to grow and survive, and so, little is known about the effects of low temperatures on genes related to food intake and inflammatory responses. This study brought insights about the modulation of genes in different tissues of Nile tilapia chronically exposed to low temperatures. Thus, sixty animals were divided in two experimental groups: a control group in which the animals remained at the optimum temperature of 24 °C; and an exposed to cold group, in which a decrease in the water temperature was applied until reaching 15 °C. These conditions were maintained for 28 days. Blood samples were collected for flow cytometry analysis, while brain, spleen, liver, and kidney tissues were collected for total RNA extraction, followed by quantitative PCR (RT-qPCR). For genes related to feeding process pathway, it was observed an upregulation in pyy and a downregulation of npy and cart gene expression. Also, pro-inflammatory cytokine genes were modulated in the spleen, kidney and liver with a higher expression of il-1b and tnfα and a reduction in the il-8 and nf-κß gene expressions in the group exposed to 15 °C. The fish exposed to cold presented higher serum cortisol levels than the ones from control group. The blood cell analysis showed a lower level of membrane fluidity and a higher DNA fragmentation and cell disruption in the group exposed to cold. These findings suggest an important effect of a stressful situation in the tilapia organism due to cold exposure. This study brings insights on tilapia wellbeing under low temperature stress. It can be a first step to understanding the appropriate way to cope with cold impacts on aquaculture.

Nanocarried antioxidants in freezing extenders for boar spermatozoa.

Post-thawing cryoinjuries in boar spermatozoa due to oxidative stress may be reduced by adding nanoencapsulated antioxidants to freezing extenders. This study evaluated post-thawing kinetics, structural and biochemical functions of boar spermatozoa frozen with extenders including resveratrol and vitamin E loaded into polymeric nanocapsules. Resveratrol was added at 0 (control), 5, 10, 20, 40 and 80 µg/ml, whereas Vitamin E was added at 0 (control), 50, 100, 200 and 400 µg/ml. Both antioxidants were tested in free and nanoencapsulated presentations. In contact with empty nanocapsules, some sperm kinetics parameters were impaired compared to the control (p < .05), whereas lipoperoxidation declined (p < .05). With inclusion of 40 µg/ml nanoencapsulated resveratrol, some sperm kinetics parameters were improved (p < .01), but sperm motility, structural and biochemical functions did not differ from the control (p > .05). No improvement in sperm quality occurred with inclusion of vitamin E, although sperm kinetics with 400 µg/ml nanoencapsulated vitamin E was reduced compared to the control (p < .01). Inclusion of 40 µg/ml nanoencapsulated resveratrol benefitted boar sperm kinetics after thawing, but no improvement resulted from inclusion of vitamin E.

Efficiency and cell viability implications using tip type electroporation in zebrafish sperm cells.

Sperm-mediated gene transfer (SMGT) has a potential use for zebrafish transgenesis. However, transfection into fish sperm cells still needs to be improved. The objective was to demonstrate the feasibility of tip type electroporation in zebrafish sperm, showing a protocol that provide high transfection efficiency, with minimal side-effects. Sperm was transfected with a Cy3-labelled DNA using tip type electroporation with voltages ranging from 500 to 1500 V. Sperm kinetics parameters were assessed using Computer Assisted Semen Analysis (CASA) and cell integrity, reactive oxygen species (ROS), mitochondrial functionality and transfection rate were evaluated by flow cytometry. The transfection rates were positively affected by tip type electroporation, reaching 64.9% ± 3.6 in the lowest voltage used (500 V) and 86.6% ± 1.9 in the highest (1500 V). The percentage of overall motile sperm in the electrotransfected samples was found to decrease with increasing field strength (P < 0.05). Increase in the sperm damaged plasma membrane was observed with increasing field strength (P < 0.05). ROS and sperm mitochondrial functionality did not present a negative response after the electroporation (P > 0.05). Overall results indicate that tip type electroporation enhances the internalization of exogenous DNA into zebrafish sperm cells with minimal harmful effects to sperm cells.

DNA microarray for forensic intelligence purposes: High-density SNP profiles obtained directly from casework-like samples with and without a DNA purification step.

SNP analyses from a forensic intelligence perspective have proven to be an important tool to restrict the number of suspected offenders and find missing persons. DNA microarray assays have been demonstrated as a potential feature in forensic analysis, like such as forensic genetic genealogy. The objective of this study was to describe the results from DNA microarray assay from saliva samples deposited on a glass surface collected from by a double swab technique, commonly applied in crime scenes. Eighteen samples from the same person were subjected to Infinium® Global Screening Array-24 v1.0 (~642.824 SNP markers) in two different protocols - with or without the DNA purification procedure. The measured genotype was compared with a Consensus Genotype, obtained from standard control samples, and the parameters such as Call Rate and GenCall Scores were evaluated. Results showed that the Call Rate parameter is enough to estimate the probability of obtaining a correct genotype in the SNP assay. Reliable genotypes with a confidence level of more than 90% (at least 90.15%) were observed in Call Rates above 69.41%, regardless of the experimental condition. Our data demonstrate that DNA Microarray from samples collected under conditions such as those found at crime scenes can generate high-density SNP genetic profiles with a confidence level greater than 90%. Enzymatic adjustments and protocol changes may enable DNA microarray assays for crime analysis and investigation purposes eliminating the purification step in the future. Our data suggest that DNA microarray can support criminal investigation teams from a forensic intelligence perspective.

Exposure to salinity induces oxidative damage and changes in the expression of genes related to appetite regulation in Nile tilapia (Oreochromis niloticus).

Variations in water salinity and other extrinsic factors have been shown to induce changes in feeding rhythms and growth in fish. However, it is unknown whether appetite-related hormones mediate these changes in Nile tilapia (Oreochromis niloticus), an important species for aquaculture in several countries. This study aimed to evaluate the expression of genes responsible for appetite regulation and genes related to metabolic and physiological changes in tilapia exposed to different salinities. Moreover, the study proposed to sequence and to characterize the cart, cck, and pyy genes, and to quantify their expression in the brain and intestine of the fish by quantitative polymerase chain reaction (qPCR). The animals were exposed to three salinities: 0, 6, and 12 parts per thousand (ppt) of salt for 21 days. Furthermore, lipid peroxidation, reactive oxygen species, DNA damage, and membrane fluidity in blood cells were quantified by flow cytometry. The results indicated an increased expression of cart, pyy, and cck and a decreased expression of npy in the brain, and the same with cck and npy in the intestine of fish treated with 12 ppt. This modulation and other adaptive responses may have contributed to the decrease in weight gain, specific growth rate, and final weight. In addition, we showed oxidative damage in blood cells resulting from increasing salinity. These results provide essential data on O. niloticus when exposed to high salinities that have never been described before and generate knowledge necessary for developing biotechnologies that may help improve the production of economically important farmed fish.

Delivery of cloned offspring: experience in Zebu cattle (Bos indicus).

The production of a healthy cloned calf is dependent on a multitude of successful steps, including reprogramming mediated by the oocyte, the development of a functional placenta, adequate maternal-fetal interaction, the establishment of a physiological metabolic setting and the formation of a complete set of well-differentiated cells that will eventually result in well-characterised and fully competent tissues and organs. Although the efficiency of nuclear transfer has improved significantly since the first report of a somatic cell nuclear transfer-derived animal, there are many descriptions of anomalies concerning cloned calves leading to high perinatal morbidity and mortality. The present article discusses some our experience regarding perinatal and neonatal procedures for cloned Zebu cattle (B. indicus) that has led to improved survival rates in Nellore cloned calves following the application of such 'labour-intensive technology'.

Safety assessment of poly(N-vinylcaprolactam) as a potential drug carrier in extenders for boar sperm cryopreservation.

Polymers may be used to deliver compounds in freezing extenders to minimize injuries in spermatozoa during cryopreservation, although their activity and toxicity for boar sperm are unknown. This study investigated the effects of the polymer (N-vinylcaprolactam) (PNVCL), when included in extenders for boar sperm cryopreservation. In Experiment 1, sperm was exposed to PNVCL at: 0 (control); 39.1; 78.1; 156.3; and 312.5 µg/mL. Spermatozoa structure, kinetics and biochemical functions were unaltered in contact with PNVCL at 38 °C (P > .05) but declined with prolonged exposure (10, 60 and 120 min) in all treatments (P > .05). In Experiment 2, after inclusion of PNVCL in the freezing extender at the same concentrations, post-thawing sperm quality did not differ compared to the control (P > .05). Lipid peroxidation and the production of reactive oxygen species were the only parameters of sperm quality that were unaffected in both experiments, even after contact with PNVCL for 120 min (P > .05). As no negative effects were observed in post-thawing boar sperm quality, PNVCL did not incur in cytotoxicity and may be a potential carrier for antioxidants in freezing extenders.

Evaluation of qPCR reference genes in GH-overexpressing transgenic zebrafish (Danio rerio).

Reference genes (RGs) must have a stable expression in tissues in all experimental conditions to normalize real-time quantitative reverse transcription PCR (qRT-PCR) data. F0104 is a highly studied lineage of zebrafish developed to overexpress the growth hormone (GH). It is assumed that the transgenic process may influence the expression levels of commonly used RGs. The objective of the present study was to make a comprehensive analysis of stability of canditade RGs actb1, actb2, b2m, eif2s2, eef1a1, gapdh, rplp2, rpl7, rpl13α, tuba1, and rps18, in gh-transgenic and non-transgenic zebrafish. Liver, brain, intestine and muscle samples from both groups had qRT-PCR results analyzed by dCt, geNorm, NormFinder, BestKeeper, and RefFinder softwares. Consensus analyses among software concluded that rpl13α, rpl7, and eef1a1 are the most stable genes for zebrafish, considering the studied groups and tissues. Gapdh, rps18, and tuba1 suffered variations in stability among different tissues of both groups, and so, they were listed as the genes with lowest stability. Results from an average pairwise variations test indicated that the use of two RGs would generate reliable results for gene expression analysis in the studied tissues. We conclude that genes that are commonly used in mammals for qRT-PCR assays have low stability in both non-transgenic and gh-transgenic zebrafish reinforcing the importance of using species-specific RGs.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome.

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Voltages up to 600V did not affect cryopreserved bovine spermatozoa on capillary-type electroporation.

Physical methods such as electroporation have been used to improve the DNA uptake efficiency of sperm cells. This study aims to develop an efficient capillary-type electroporation method for incorporation of exogenous DNA into bovine cryopreserved sperm cells with minimal detrimental effects for later use in SMGT. Electroporation of the samples was performed in 2 different groups (with 1 µg of DNA and without DNA transfection) and under five different voltages: 500 V, 600 V, 700 V, 800 V and 900 V. Non-electroporated sperm cells (with and without DNA) were used as control. Kinetics parameters were determined using computer assisted semen analyses, whereas membrane integrity, fluidity, mitochondrial function and DNA uptake were evaluated by flow cytometry. Results revealed that all tested voltages reduced electroporated sperm motility (P < 0.05) when compared to the control (non-electroporated cells). Mitochondrial function results showed no statistical difference among groups. Similarly, groups electroporated with lower (500 V, 600 V and 700 V) voltages showed no difference in cell membrane integrity and fluidity. Groups electroporated at higher voltages (800 V and 900 V) demonstrated negative effects in cells membrane integrity when compared to other groups and control. Also, all electroporated groups demonstrated significant higher percentages of transfected sperm cells when compared to the control group (P < 0.05). Under the recommendation of using voltages up to 600 V, this method represents a safe and efficient alternative for electroporation of bovine spermatozoa.

Exposure of Somatic Cells to Cytoplasm Extracts of Porcine Oocytes Induces Stem Cell-Like Colony Formation and Alters Expression of Pluripotency and Chromatin-Modifying Genes.

Cell permeabilization followed by exposure to cytoplasmic extracts of oocytes has been proposed as an alternative to transduction of transcription factors for inducing pluripotency in cultured somatic cells. The main goal in this study was to investigate the effect of treating porcine fibroblast cells with cytoplasmic extracts of GV-stage oocyte (OEx) followed by inhibition of histone deacetylases with Scriptaid (Scrip) on the formation of stem cell-like colonies and expression of genes encoding pluripotency and chromatin-modifying enzymes. Stem cell-like colonies start developing ∼2 weeks after treatment in cells exposed to OEx or OEx + Scrip. The number of cell colonies at the first day of appearance and 48 hours later was also similar between OEx and OEx + Scrip treatments. Transcripts for Nanog, Rex1, and c-Myc genes were detected in most cell samples that were analyzed on different days after OEx treatment. However, Sox2 transcripts were not detected and only a small proportion of samples had detectable levels of Oct4 mRNA after OEx treatment. A similar pattern of transcripts for pluripotency genes was observed in cells treated with OEx alone or OEx + Scrip. Transcript levels for Dnmt1 and Ezh2 were reduced at Day 3 after treatment in cells exposed to OEx. These findings revealed that: (a) exposure to OEx can induce a partial reprogramming of fibroblast cells toward pluripotency, characterized by colony formation and activation of pluripotency genes; and (b) inhibition of histone deacetylases does not improve the reprogramming effect of OEx treatment.

Binding pattern and toxicological effects of lectins from genus Canavalia on bovine sperm.

The aim of this study was to determine the binding patterns of Canavalia ensiformis (ConA), Canavalia boliviana (ConBol) and Canavalia brasiliensis (ConBr) lectins to bovine sperm and their effects on sperm motility, viability, lipid peroxidation, reactive oxygen species production and fertilization ability. ConA bound to whole spermatozoa, with the exception of the equatorial segment, ConBol did not interact with the acrosome region and ConBr exhibited a fragmented binding pattern. The three lectins decreased sperm motility but did not affect cell viability or lipid peroxidation. Nevertheless, ROS production was increased in comparison to controls and a reduction in the cleavage and blastocyst ratio was induced in comparison to controls. In conclusion, this study determined that structurally similar lectins interact differently with bovine sperm and affect sperm motility, viability, lipid peroxidation, ROS production and fertilization ability in various ways.