Exogenous DNA uptake by South American catfish (Rhamdia quelen) spermatozoa after seminal plasma removal

The effects of the replacement of bread yeast by soybean meal in diets for Rhamdia quelen larvae on growth, survival, and intestinal morphology were analysed. Larvae were fed for 20 days with five diets: a control diet formulated with 57% of bread yeast, and other four diets in which soybean meal at concentrations of 14.25, 28.5, 42.75 and 57% was added to obtain 25, 50, 75 and 100% of bread yeast replacement. Growth and survival parameters were negatively affected by dietary soybean meal inclusion. Larvae fed control diet showed significantly higher mean weight, specific growth rate, final biomass, and survival rate than larvae from other treatments. Enterocyte height and fold width of the posterior intestine showed highest values in the control group, and an inverse linear relationship with the level of dietary soybean meal inclusion was observed, however, in the anterior intestine the morphology parameters were not affected by the diet. These results indicate that inclusion of soybean meal in diets for R. quelen larvae negatively affects growth and survival, as well as the capacity for digestion and absorption of nutrients, mainly in the posterior intestine.

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The Helicobacter pylori HpyAXII restriction–modification system limits exogenous DNA uptake by targeting GTAC sites but shows asymmetric conservation of the DNA methyltransferase and restriction endonuclease components

Nucleic Acids Research ◽

10.1093/nar/gkn718 ◽

2008 ◽

Vol 36 (21) ◽

pp. 6893-6906 ◽

Cited By ~ 39

Author(s):

Olivier Humbert ◽

Nina R. Salama

Keyword(s):

Helicobacter Pylori ◽

Restriction Endonuclease ◽

Dna Methyltransferase ◽

Dna Uptake ◽

Modification System ◽

Exogenous Dna ◽

Restriction Modification System ◽

Restriction Modification

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Comparison of different methods for exogenous DNA uptake by bovine spermatozoa

Brazilian Journal of Veterinary Research and Animal Science ◽

10.11606/issn.1678-4456.v52i1p78-86 ◽

2015 ◽

Vol 52 (1) ◽

pp. 78 ◽

Cited By ~ 2

Author(s):

Renata Simões ◽

Weber Beringui Feitosa ◽

Marcella Pecora Milazzotto ◽

Alessandra Corallo Nicacio ◽

Flavia Regina Oliveira de Barros ◽

...

Keyword(s):

Dna Uptake ◽

Exogenous Dna ◽

De Se ◽

Bovine Spermatozoa

<p>Apesar da manipulação genética de animais domésticos ser de grande interesse para a produção animal e para a indústria farmacêutica, a sua eficiência ainda é insatisfatória. A injeção pronuclear, a técnica mais utilizada para tal proposito, principalmente em camundongos, ainda apresenta limitações para esta espécie. Algumas alternativas têm sido desenvolvidas como o uso de espermatozoides como vetores para transferência genica, na qual a célula espermática tem habilidade espontânea de se ligar a molécula de DNA e internaliza-la. Dado o potencial da transferência genica mediada por espermatozoide para animais domésticos transgênicos, o objetivo do presente trabalho foi a avaliação de quatro métodos de incorporação de DNA para a transferência genica mediada por espermatozoides na espécie bovina: incubação com DNA, alteração da membrana plasmática induzida por cálcio ionóforo seguida por incubação com o DNA exógeno, eletroporação e lipofecção. Espermatozoides não expostos ao DNA exógeno foram usados como grupo controle. Os índices de clivagem, blastocisto e eclosão foram avaliados, respectivamente, as 72 horas após a inseminação dos oócitos, bem como, aos 9 e 12 dias de cultivo embrionário. Os embriões positivos para o DNA exógeno foram avaliados por PCR. Nenhum efeito de tratamento foi observado nos índices de clivagem, blastocisto e eclosão. Além disso, a porcentagem de blastocistos positivos para o DNA exógeno não diferiu entre os grupos experimentais. Apesar do baixo número de embriões positivos para DNA exógeno, os resultados obtidos mostram que todos os tratamentos apresentaram eficiências similares. A conclusão obtida foi que, apesar de os índices de desenvolvimento embrionário terem sido similares e constante em todos os grupos experimentais, outros fatores como a sequência, o tamanho e a concentração do DNA exógeno devem ser avaliados para melhorar a transferência genica mediada por espermatozoides.</p>

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Activity patterns of South American silver catfish (Rhamdia quelen)

Brazilian Journal of Biology ◽

10.1590/s1519-69842006000300024 ◽

2006 ◽

Vol 66 (2a) ◽

pp. 565-574 ◽

Cited By ~ 22

Author(s):

U. H. Schulz ◽

C. Leuchtenberger

Keyword(s):

Woody Debris ◽

Activity Patterns ◽

Swimming Activity ◽

Fish Length ◽

South American ◽

Silver Catfish ◽

Frequent Use ◽

Rhamdia Quelen ◽

Lakes And Reservoirs ◽

The Mean

The South American silver catfish (Rhamdia quelen) is a widely distributed species in Central and South America in areas east of the Andes between Venezuela and the northern parts of Argentina. The bottom dwelling species occurs in lakes and reservoirs as well as in rivers. Between June 2000 and December 2001, sixteen silver catfish were tracked during fourteen 24-h cycles in two-hour-intervals, with the aim of investigating daily movements and habitat use. Covered distances varied between 0 m/2 h and 326 m/2 h and the mean distance covered in 2 h was 25.6 m. The mean activity of individual silver catfish varied between 5.6 m/2 h and 81.4 m/2 h. The swimming activity was linearly related to the total fish length. The highest mean swimming activity occurred in the morning and at nightfall. Silver catfish concentrated in three areas of frequent use. All of them were characterized by steep banks providing shelter in the form of rip-rap or large woody debris. Vertically, silver catfish preferred the upper 2 m layer where tracked fish encountered higher temperatures and higher dissolved oxygen concentrations.

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267 EFFECT OF SEMINAL PLASMA IN LAMA GLAMA SPERM

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab267 ◽

2015 ◽

Vol 27 (1) ◽

pp. 223 ◽

Cited By ~ 8

Author(s):

M. Carretero ◽

F. Fumuso ◽

M. Miragaya ◽

C. Herrera ◽

S. Giuliano

Keyword(s):

Sperm Motility ◽

Seminal Plasma ◽

Membrane Integrity ◽

South American ◽

South American Camelids ◽

Progressive Motility ◽

Coomassie Blue ◽

Blue Stain ◽

Lama Glama ◽

Motility Of Sperm

In South American camelids, raw semen only presents sperm with oscillatory movements. Therefore, it is necessary to treat these cells to enable them to acquire progressive motility. The effects of raw seminal plasma (SP) on sperm movement patterns (oscillatory, progressive, and hyperactive) have apparently not yet been reported. The objective of this study was to determine effects of raw seminal plasma on sperm motility, viability, and acrosomal status in fresh llama semen. A total of 15 ejaculates were collected (electroejaculation) from 5 llamas (n = 5, r = 3). Each ejaculate was diluted 4 : 1 in 0.1% collagenase in HEPES-TALP (HT) medium and incubated 4 min at 37°C, with the objective of separating spermatozoa from SP. Immediately after incubation, each ejaculate was divided into 2 and centrifuged for 8 min at 800 × g. Pellets were resuspended in either HT or raw SP and maintained at 37°C until evaluation (at 0, 1.5, and 3 h). Sperm motility was evaluated using a phase contrast microscope and a warm stage. Propidium iodide and carboxyfluorescein diacetate were used for assessing membrane integrity (viability). Acrosomal status was evaluated with the Coomassie blue stain. A split-plot design was used with treatment as a factor, with 2 levels (HT and SP) and time as the other factor, with 3 levels (0, 1.5, and 3 h), and blocked by males. There was no significant interaction between treatments (HT and SP) and times (0, 1.5, and 3 h) for each of the seminal characteristics evaluated. Progressive sperm motility was observed after collagenase treatment in all samples. Progressive motility disappeared immediately after the addition of raw SP and showed only oscillatory movements. In contrast, samples incubated in HT maintained progressive motility and became hyperactive. There were no differences (P > 0.05) in total motility of sperm incubated in HT among incubation times (0 h: 30.8 ± 18.9%; 1.5 h: 26.5 ± 11.5%; and 3 h: 21.5 ± 13.5%). However, in samples incubated with SP, a decrease (P < 0.05) in total sperm motility was detected after 3 h of incubation (0 h: 16.5 ± 12.6%, 3 h: 2.3 ± 3.2%). Sperm viability was not different (P > 0.05) between treatments (HT and SP); samples incubated in HT retained 78.4% of the initial viability (32.8/41.8, 3 h/0 h), and samples incubated in SP retained 69.7% of their initial viability (24.4/35.0, 3 h/0 h). The percentage of spermatozoa with intact acrosomes was not different (P > 0.05) between treatments (HT and SP); however, the percentage of sperm with intact acrosomes decreased after 3 h of incubation in both samples (HT and SP). Due to the presence of a high percentage of progressive and hyperactive motile sperm in samples incubated in HT and their absence in samples incubated in SP, we concluded that raw seminal plasma preserved oscillatory sperm motility. Further studies are needed to understand the effects of SP on South American camelid spermatozoa.

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404 DNA TAKE-UP BY SPERM AND IN VITRO EMBRYO DEVELOPMENT BY SPERM-MEDIATED GENE TRANSFER IN THE PIG

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab404 ◽

2007 ◽

Vol 19 (1) ◽

pp. 317

Author(s):

T. S. Kim ◽

Y. Cao ◽

H. T. Cheong ◽

B. K. Yang ◽

C. K. Park

Keyword(s):

Gene Transfer ◽

Transfection Efficiency ◽

The Other ◽

Control Group ◽

Dna Uptake ◽

Alkaline Lysis ◽

Exogenous Dna ◽

Other Hand ◽

Dna Complex

Sperm mediated gene transfer (SMGT) is based on the ability of spermatozoa to bind and internalize exogenous DNA and transfer it into the oocytes at fertilization. The purpose of this study was to assess introducing exogenous DNA into boar spermatozoa by DNA solution or DNA/liposome complex under different conditions (period of incubation, exogenous DNA, liposome, and concentration of spermatozoa). Genomic DNA of sperm loaded with DNA by treatment was isolated by alkaline lysis. Quantitation of exogenous DNA amplified by PCR was analyzed by agarose electrophoresis densitometry. The quality of treated spermatozoa under the best conditions or no treatment (control) was evaluated during incubation (0, 2, 4, and 6 h) for viability (SYBR-14/PI), motility (Makler counting chamber), morphology (rose bengal staining), and acrosomal status (Coomassie staining). Sperm loaded with DNA also were used for in vitro fertilization. Immature oocytes incubated in TCM-199 medium for 44 h were fertilized in mTBM medium for 6 h and cultured in PZM-3. Cleavage and development of embryos were assessed on Days 2 and 7 of culture, respectively. Transfection rates at the blastocyst stage were assessed by PCR analysis. Data were evaluated by Duncan's multiple-range test using the GLM procedure. In the preliminary experiment, DNA uptake of spermatozoa by DNA solution and liposome/DNA complex was completed within 90-120 min. Transfection efficiency of spermatozoa was significantly (P < 0.05) higher in the 105 spermatozoa group than in the other groups (104, 106, and 107 spermatozoa). The transfection efficiency was gradually increased by increasing the concentration of exogenous DNA. On the other hand, viability of transfected spermatozoa by all treatments (control, DNA solution, and DNA/liposome) at 0 h (72.3 � 0.2, 70.8 � 1.8, and 68.0 � 2.2%, respectively) of storage was significantly (P < 0.05) lower than for fresh spermatozoa (83.3 � 1.7%). Survival and motility of all treatments after 4 h of storage were significantly (P < 0.05) lower than at 0 and 2 h. Both abnormality and acrosome reaction of spermatozoa were gradually increased with prolonged storage periods. On the other hand, the cleavage rate of embryos by DNA/liposome complex (56.3 � 2.3%) was significantly (P < 0.05) lower compared to both DNA solution (64.0 � 1.1%) and control (67.8 � 2.3%). The developmental rates of blastocysts were significantly (P < 0.05) lower in the liposome/DNA complex and DNA solution groups (9.1 � 1.3 and 11.3 � 0.8%) than in the control group (22.2 � 0.6%). The transfection rates of blastocysts were higher in the liposome/DNA group (54.3 � 12.0%) than in the DNA solution group (38.7 � 6.6%). These results show that the SMGT method under the control conditions efficiently transfers exogenous DNA into the porcine oocytes. This work was supported by the Research on the Production of Bio-organs (No. 2005 03020302) Ministry of Agriculture and Forestry, Republic of Korea

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Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1998.275.3.l452 ◽

1998 ◽

Vol 275 (3) ◽

pp. L452-L460 ◽

Cited By ~ 7

Author(s):

A. Keith Tanswell ◽

Olivier Staub ◽

Richard Iles ◽

Rosetta Belcastro ◽

Judy Cabacungan ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Reporter Gene ◽

Fetal Lung ◽

Reporter Gene Expression ◽

Lung Epithelial Cells ◽

Dna Uptake ◽

Exogenous Dna ◽

Lipid Ratio ◽

Lung Epithelial

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl- sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 μM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 μM chloroquine.

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The Bacillus Subtilis K-State Promotes Stationary-Phase Mutagenesis via Oxidative Damage

Genes ◽

10.3390/genes11020190 ◽

2020 ◽

Vol 11 (2) ◽

pp. 190

Author(s):

Holly A. Martin ◽

Amanda A. Kidman ◽

Jillian Socea ◽

Carmen Vallin ◽

Mario Pedraza-Reyes ◽

...

Keyword(s):

Bacillus Subtilis ◽

Oxidative Damage ◽

Stationary Phase ◽

Bacterial Cells ◽

Dna Uptake ◽

Cellular Division ◽

Exogenous Dna ◽

K Cells ◽

Induced Mutagenesis ◽

Underlying Mechanisms

Bacterial cells develop mutations in the absence of cellular division through a process known as stationary-phase or stress-induced mutagenesis. This phenomenon has been studied in a few bacterial models, including Escherichia coli and Bacillus subtilis; however, the underlying mechanisms between these systems differ. For instance, RecA is not required for stationary-phase mutagenesis in B. subtilis like it is in E. coli. In B. subtilis, RecA is essential to the process of genetic transformation in the subpopulation of cells that become naturally competent in conditions of stress. Interestingly, the transcriptional regulator ComK, which controls the development of competence, does influence the accumulation of mutations in stationary phase in B. subtilis. Since recombination is not involved in this process even though ComK is, we investigated if the development of a subpopulation (K-cells) could be involved in stationary-phase mutagenesis. Using genetic knockout strains and a point-mutation reversion system, we investigated the effects of ComK, ComEA (a protein involved in DNA transport during transformation), and oxidative damage on stationary-phase mutagenesis. We found that stationary-phase revertants were more likely to have undergone the development of competence than the background of non-revertant cells, mutations accumulated independently of DNA uptake, and the presence of exogenous oxidants potentiated mutagenesis in K-cells. Therefore, the development of the K-state creates conditions favorable to an increase in the genetic diversity of the population not only through exogenous DNA uptake but also through stationary-phase mutagenesis.

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Embryonic and larval development of Jundiá (Rhamdia quelen, Quoy & Gaimard, 1824, Pisces, Teleostei), a South American Catfish

Brazilian Journal of Biology ◽

10.1590/s1519-69842006000600013 ◽

2006 ◽

Vol 66 (4) ◽

pp. 1057-1063 ◽

Cited By ~ 28

Author(s):

C. R. Pereira ◽

L. J. G. Barcellos ◽

L. C. Kreutz ◽

R. M. Quevedo ◽

F. Ritter ◽

...

Keyword(s):

South American ◽

Fish Production ◽

Perivitelline Space ◽

Southern South America ◽

Water Influx ◽

Hormone Injection ◽

Toxicological Studies ◽

Rhamdia Quelen ◽

Cleavage Stages ◽

Embryonic And Larval Development

The jundiá (Rhamdia quelen, Quoy & Gaimard) is an endemic South American fish species. Because this species supports cold winters and grows faster during warm months, it has begun to be viewed as an ideal species for fish production in southern South America. In the present study, jundiá oocytes used were obtained by extrusion from females after hormone injection. Soon after hydration, the eggs were transferred to 50 L conic glass incubators, with constant and controlled water influx. Samples of fertilized eggs were transferred to Petri dishes and, examined under a stereoscopic microscope, were spherical, demersal, and non-adhesive with defined perivitelline space and resistant chorion. Cleavage stages occurred during the first 3.5 h. After hatching, larvae were transferred to 200 L glass fiber incubators. First signs of embryo movement were observed 21 h after fertilization; larval eclosion occurred 30.5 h after fertilization. Present findings may provide a basis for studies aimed at determining the complete ontogeny of jundiá and may be useful in eco-toxicological studies.

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