ABSTRACT
Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).
Subject(s)
Animals , Female , Male , Mice , Benzimidazoles/chemistry , Cryopreservation/veterinary , Embryo Transfer/veterinary , Fertilization in Vitro/veterinary , Fluorescent Dyes/chemistry , Mice, Transgenic , Microscopy, Fluorescence/methods , Propidium/chemistry , Semen Analysis/methods , Semen Preservation/veterinary , Spermatozoa/physiologyABSTRACT
Supplementation of beta-mercaptoethanol (beta-ME) in in vitro maturation (IVM) medium was shown to improve embryo development and quality in several species. Epidermal growth factor (EGF) was also shown to improve IVM of human oocyte and embryo development after in vitro fertilization (IVF). The effect of these two compounds were suggested to be mediated through the synthesis of glutathione (GSH) which is known to play an important role in protecting the cell or embryos from oxidative damage. Thus, it is suggested that supplementation of canine IVM medium with beta-ME or EGF may be of benefit due to its positive role in IVM of various mammalian oocytes and embryo development, including cattle, pigs, rodents and humans. This study investigates the effect of ovarian estrus stage on canine oocyte quality and supplementation of medium with beta-ME or EGF on IVM of canine oocytes. As results, a significantly higher percentage of oocytes progressed to metaphase II (MII) stage in 50 or 100 microM of beta-ME supplemented oocytes collected from the follicular stage. The maturation rate to metaphase I (MI) stage was also significantly higher in oocytes collected from follicular stage and cultured with 25 or 100 microM compared to other experimental groups. After IVM culture, oocytes recovered from dogs with the follicular stage and matured in TCM-199 supplemented with 20 ng/ml EGF yielded better oocyte maturation to MII phase compared to other groups. Taken together, supplementation of beta-ME (50 or 100 microM) or EGF (20 ng/ml) improved IVM of canine oocytes to MII stage.
Subject(s)
Animals , Female , Benzimidazoles/chemistry , Dogs/physiology , Epidermal Growth Factor/pharmacology , Estrus/physiology , Fluorescent Dyes/chemistry , Meiosis/drug effects , Mercaptoethanol/pharmacology , Microscopy, Ultraviolet/veterinary , Oocytes/drug effects , Ovary/drug effectsABSTRACT
Benzimidazo [1, 2-a] benzimadozoles [1, R=H] were synthesized by the trialkylphosphite-induced deoxygenation and thermolysis of 1-0-nitrophenyl and 1-0-azidophenyl benzimidazole. Spectral and other properties of the product and the intermediate compounds are reported
Subject(s)
Benzimidazoles/chemistryABSTRACT
Se describe la síntesis de derivados nucleosídicos acíclicos 1-sustituidos del 5(6)-bencimidazol y su evaluación farmacológica como compuestos antivirales. Los compuestos fueron sintetizados utilizando 5(6)-nitrobencimidazol como material de partida con los alcoholes sustituidos, clorometilados apropiados, con el fin de obtener análogos de Acyclovir y Ganciclovir, compuestos antivirales conocidos. La actividad antiviral de estos compuestos fue evaluada utilizando cepas de Herpes simplex 1 y Polivirus 1 y bajo las condiciones ensayadas resultaron inactivos