Effect of glutaraldehyde concentration and fixative temperature on the number of spermatozoa with normal acrosomes in goat semen

Two experiments were conducted to determine the best fixative solution and the most suitable temperature for fixing sperm cells from goat ejaculates. In Experiment 1, a 6×3 factorial design was used to test 4 glutaraldehyde concentrations (0.5, 1, 2 and 4%) plus 1 treatment that did not contain a fixative (0%) but to which 0.3% sodium fluoride (NaF) had been added to immobilize the spermatozoa; the control treatment contained no fixative or NaF. The 6 treatments were tested with 3 different solvents PBS, Na citrate and BL-1, representing a total of 18 samples per replicate. The fixed samples always provided a significantly higher (P<0.01) percentage of normal acrosomes than the unfixed samples, whether immobilized with NaF or observed inmediately after dilution. In Experiment 2, a 3×3 factorial design was used to determine the effect of the temperature of the glutaraldehyde fixative solution on the number of morphologically normal acrosomes from goat semen samples kept at 3 different temperatures. Our findings indicated that at all 3 fixative solution temperatures (5, 20 and 37°C) there was a significant difference (P<0.01) in the percentage of normal acrosomes. At 5°C, glutarhaldehyde yielded a general mean number of 53.6 normal acrosomes vs 75.1 at 20°C and 83.05 at 37°C. Based on these results, we recommend that the temperature of a fixative solution be established when designing an experiment using goat semen, since the temperature has a significant effect on the number of the normal acrosomes found in a semen sample. © 1992

Transformación de espermatogenias tipo A de ratón medida por liposomas

La transformación de cspcrnatogonias indiferenciadas empieza a ser un sistema alternativo para la modifi-cación <le la línea germinal animal y obtención de animales genéticamente modificados a raíz (lo la posibilidad de trasplante de estas células al testículo de un macho receptor de la misma o diferente especie. El desarrollo de esta metodología implica la necesidad de optimizar un sistema de cultivo in vitro que permita cl mantenimiento a largo plazo de esta estirpe celular de ¡orna indiferenciada, y también la consecución de un sistema de transfcc-ción eficiente. Este trabajo explora cl segundo aspecto comparando la capacidad de transtccción de tres diferen-tes liposomas utilizando una construcción basada en la expresión de proteína fluorescente verde (GFP) como marcador de expresión. Nuestro estudio demuestra que es posible transfectar una población celular enriquecida en cspermatogonias tipo A mediante diferentes fórmulas lipidicas (lipofcctina, lipotcctamina y ccllfcctina) con un rango bajo de citotoxicidad. Se han detectado diferencias en cl nivel de expresión de GFP en las espcrmato-gonias transfectadas dependientes del liposoma utilizado y del tiempo post-transfección. Los porcentajes de cé-lulas transfectadas más elevados se obtuvieron con lipofcctamina 48 horas después de la transfección

Transgenic mouse offspring generated by ROSI

The production of transgenic animals is an important tool for experimental and applied biology. Over the years, many approaches for the production of transgenic animals have been tried, including pronuclear microinjection, sperm-mediated gene transfer, transfection of male germ cells, somatic cell nuclear transfer and the use of lentiviral vectors. In the present study, we developed a new transgene delivery approach, and we report for the first time the production of transgenic animals by co-injection of DNA and round spermatid nuclei into non-fertilized mouse oocytes (ROSI). The transgene used was a construct containing the human CMV immediate early promoter and the enhanced GFP gene. With this procedure, 12% of the live offspring we obtained carried the transgene. This efficiency of transgenic production by ROSI was similar to the efficiency by pronuclear injection or intracytoplasmic injection of male gamete nuclei (ICSI). However, ICSI required fewer embryos to produce the same number of transgenic animals. The expression of Egfp mRNA and fluorescence of EGFP were found in the majority of the organs examined in 4 transgenic lines generated by ROSI. Tissue morphology and transgene expression were not distinguishable between transgenic animals produced by ROSI or pronuclear injection. Furthermore, our results are of particular interest because they indicate that the transgene incorporation mediated by intracytoplasmic injection of male gamete nuclei is not an exclusive property of mature sperm cell nuclei with compact chromatin but it can be accomplished with immature sperm cell nuclei with decondensed chromatin as well. The present study also provides alternative procedures for transgene delivery into embryos or reconstituted oocytes

Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa

This study analyzes the relationship between chromatin condensation and field fertility, expressed as 90 days non-return rate (NRR), of bulls actively used by AI studs. Frozen-thawed semen from five bulls (six ejaculates per bull, three straws per ejaculate), that showed a non-return rate between 60 and 80%, were analyzed to assess sperm chromatin condensation and stability. The chromatin condensation was determined by flow cytometry using propidium iodide as fluorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. Coefficient of variation among replicates was less than 7% and 5% for chromatin condensation and stability, respectively. No correlation was present between chromatin condensation and NRR. However, significant correlation was found between chromatin stability and NRR. Chromatin stability was higher (P < 0.05) in those bulls that showed higher fertility. The results obtained in this study conclude that assessment of stability could be a valuable tool for routine evaluation and identification of ejaculates with high levels of sperm chromatin abnormalities and to detect animals of higher reproductive potential. © 2004 Elsevier Inc. All rights reserved