Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage.

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.

In vitro penetration of fresh and vitrified swine oocytes by homologous spermatozoa using different incubation systems.

The present study consisted of two experiments. In the first one, ejaculates from four boars were used to compare in vitro penetration (IVP) rates of fresh and vitrified swine oocytes by homologous spermatozoa in four treatments: fresh oocytes in conventional incubation (CO2 incubator) (FC), vitrified oocytes in conventional incubation (VC), fresh oocytes in submarine (bag) incubation (FS) and vitrified oocytes in submarine incubation (VS). The IVP rates for FC, VC, FS and VS were 46.5, 44.3, 36.9 and 33.1%, respectively. Analysis through Chi-square tests identified no differences in IVP rates between FC and VC and between FS and VS (P > 0.05), but IVP rate for FC was greater (P < 0.05) than those for both FS and VS. Besides IVP rate for VC did not differ (P > 0.05) from those for FC and FS, but it was greater than that for VS (P < 0.05). Logistic regression analysis identified differential effects of treatments dependant on individual boars. The second experiment evaluated the influence of semen storage period on the semen quality of the two boars associated with greater IVP rate in the first experiment. Semen quality was estimated by IVP rate using the VC treatment and by the following methods: sperm motility, sperm morphology, hypoosmotic swelling test (HOST) and thermal stress test (TST). According to analysis using Chi-square tests, IVP rate did not differ (P > 0.05), for the first boar, between 0 (100.0%) and 24 h of semen storage (98.1%) nor after 48 and 72 h (66.0 and 59.3%, respectively), but IVP rates were greater during the 0-24 h period compared with the 48-72 h period (P < 0.05). For the second boar, IVP rate at 0 h (50.6%) was greater (P < 0.05) than at 24, 48 and 72 h of semen storage (34.3, 28.3 and 24.0%, respectively), with no further differences observed after 24 h (P > 0.05). Logistic regression analysis identified that the effect of storage on IVP rate was influenced by the effect of individual boars. No differences in semen quality during the storage period were identified by conventional methods of semen evaluation, for either boar (P > 0.05) using analysis of variance with repeated measures. These results indicate that IVP test can be used to estimate boar fertility, even when vitrified oocytes are used (if using conventional CO2 incubators) or using an alternative submarine incubation system (if using fresh oocytes). The IVP test was the only method of semen evaluation that identified the reduction in semen quality up to 72 h of storage.

Risk factors for stillbirths in two swine farms in the south of Brazil.

We evaluated stillbirth risk factors in two commercial swine farms of the Rio Grande do Sul State (south of Brazil). The study was conducted during 1 month in Farm A and during 2 months in Farm B, both during 1999. Data for all farrowings that occurred during the study period were recorded (101 for Farm A and 373 for Farm B), without interference in the farm management. In Farm A, 39% of all litters born during the period of interest had stillborn piglets and the stillborn risk for piglets was 12%. In Farm B, 25% of all litters had stillborn piglets whereas the stillborn risk was 2%. Variables considered as potential risk factors for stillbirths were: parity (1, 2-3, 4+); breed (purebred or crossbred); sow body-condition (normal or fat); use of oxytocin during parturition (yes or no); obstetric intervention through vaginal palpation (yes or no); farrowing duration (<4 or > or =4h); mummified fetuses (yes or no); total litter size (<12 or > or =12 piglets); and litter birth weight (<11 or > or =11kg). All stillborn piglets had their classification validated by necropsy. In multivariable logistic-regressions, the cases were the litters having at least one stillborn piglet. In Farm A, litters having at least 12 pigs and in which oxytocin was used during the parturition had 20.8-times-higher odds of stillborn occurrence. In Farm B, litters from sows having parity > or =4 had 2.2-times-higher odds of stillborn occurrence than litters from parity 2 to 3 females, litters having > or =12 pigs had 2.0-times-higher odds of a stillborn piglet than smaller litters and farrowings in which vaginal palpation was performed had 8.0-times-higher odds. Farrowing room management to minimize stillborn risk should target higher-parity females, large litters and optimization of practices of obstetric interventions.

Neuropeptide Y gene expression around meal time in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (-2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P less than 0.05) and significantly reduced 2 h later (P less than 0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P less than 0.05) at -2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.

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

Sperm from different species shows biological differences, determining the success or failure of the sperm-mediated gene transfer (SMGT) technique. There is evidence that exogenous DNA uptake by the spermatozoa is a species-specific and highly regulated phenomenon. Problems involving SMGT procedures might be related to activation of defenses in spermatozoa and in seminal plasma such as DNase enzymes. The objective in the present study was to transfect South American catfish spermatozoa after seminal plasma removal. Seminal plasma had a strong DNase activity that is reduced after sperm washes in isosmotic solution, in which Western blot analysis demonstrated a reduction in the DNase content after washes and Southern blot evaluations show the presence of plasmid after sperm washes. The seminal plasma DNase digests exogenous DNA in a few minutes and has an optimal activity at 43°C. Also, EDTA at 30 mM concentration inhibits the DNase activity. Using PCR the pEGFP vector was internalized by sperm cells even at lesser concentrations (5-40 ng/10(6) spermatozoa) without motility loss after seminal plasma removal. Conversely, using greater pEGFP concentrations (100 ng/10(6) spermatozoa), there were no motile cells, suggesting toxicity of exogenous DNA for sperm cells. These results are interpreted to provide information that can improve the protocol for generation of transgenic South American catfish.

Identification, tissue distribution and evaluation of brain neuropeptide Y gene expression in the Brazilian flounder Paralichthys orbignyanus.

Neuropeptide Y (NPY) is one of the most potent stimulants of food intake in vertebrates, mammals and fish. However, the present knowledge about feeding behaviour in fish is still limited and based on studies in a few species. The Brazilian flounder Paralichthys orbignyanus is being considered for aquaculture, and it is important to understand the mechanisms regulating feeding in order to improve its performance in captivity. The objectives of this study were to clone NPY cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain NPY expression to associate food intake with NPY expression levels. A 597 bp NPY cDNA was cloned from Brazilian flounder brain. NPY expression was detected in all the peripheral tissues analysed. No significant differences were observed in brain NPY gene expression over 24 h after food intake at a temperature of 15 +or- 3 degrees C. No correlation was observed among plasma glucose, total protein, cholesterol, triglycerides and NPY expression levels during this 24 h period. On the other hand, mRNA levels were increased after two weeks of fasting at elevated temperatures. Our results suggest that NPY mRNA levels in Brazilian flounder are affected by temperature.

Testis-mediated gene transfer in mice: comparison of transfection reagents regarding transgene transmission and testicular damage

Testis-mediated gene transfer (TMGT) has been used as in vivo gene transfer technology to introduce foreign DNA directly into testes, allowing mass gene transfer to offspring via mating. In this study, we used plasmid DNA (pEGFP-N1) mixed with dimethylsulfoxide (DMSO), N,N-dimethylacetamide (DMA) or liposome (Lipofectin) in an attempt to improve TMGT. Males receiving consecutive DNA complex injections were mated to normal females to obtain F0 progeny. In vivo evaluation of EGFP expression, RT-PCR and PCR were used to detect the expression and the presence of exogenous DNA in the progeny. We also evaluated possible testicular damage by histological procedures. PC R and RT-PCR analyses revealed that liposome and DMSO increased the rate of TMGT. Histological analyses demonstrated that repeated (4 times) injections of DNA complexes can affect spermatogenesis. DMSO was the most deleterious among the reagents tested. In this study, we detected the presence of transgene in the progeny, and its expression in blood cells. Consecutive injections of DNA complexes were associated with impaired spermatogenesis, suggesting requirement of optimal conditions for DNA delivery through TMGT.