A major obstacle of widespread commercial application of bovine somatic cell nuclear transfer is the low overall efficiency, that is, healthy calf-late pregnancy per transferred embryo rate. In this study, we report a series of experiments with a limited number of embryos created with handmade cloning (HMC) and transferred without or after open pulled straw vitrification. Embryo reconstruction was performed by using in vitro matured oocytes and adult ear skin fibroblasts. In two experiments, a total of 53 D7 blastocysts were developed from 188 reconstructed embryos. Fresh transfer of seven blastocysts into six recipients has resulted in three early pregnancies, two of them developed over 90 days and eventually resulted in healthy calves (33% offspring/transfer rate). In another two experiments, a total of 11 D7 blastocysts were obtained from 36 reconstructed embryos. Out of these, eight have reexpanded 18 hours after vitrification and warming. Transfer of these blastocysts into eight recipients has resulted in four early pregnancies and two live births; 25% offspring/transfer rate. These results indicate that low overall efficiency may not be an intrinsic feature of cattle cloning, and selection of the right procedures may help to overcome the actual limitations.

<p>La digestión enzimática de tejidos vivos es fundamental para la obtención de cultivos celulares primarios. El objetivo de la investigación fue evaluar el efecto de dos tratamientos enzimáticos para la producción<em> in vitro</em> de líneas celulares primarias vivas. Para ello se utilizaron biopsias obtenidas por perforación de las orejas de cuyes y conejos silvestres sometidos a digestión enzimática con colagenasa tipo I y tripsina EDTA. Los resultados indican que ambas enzimas digestivas producen cultivos celulares primarios vivos, pero la utilización de la colagenasa tipo I genera cultivos celulares primarios con mayor velocidad de crecimiento en estos animales. Los resultados podrían ayudar en la conservación<em> ex situ</em> de tejidos vivos de animales silvestres.</p><p> </p>