Desarrollo de un sistema económico para la vitrificación de embriones de conejo

[ES] Uno de los soportes más utilizados actualmente en la vitrificación de óvulos y embriones es el denominado Cryotop. Este soporte permite altos ratios de enfriamiento debido al pequeño volumen de la solución de vitrificación empleada, si bien, su precio ronda los 10 euros. El objetivo de este trabajo es comparar el Cryotop con un asa de siembra de plástico para la vitrificación de embriones de conejo. Tras la inseminación, a las 72 horas se recuperarán embriones en estadios de mórula o blastocisto temprano que serán sometidos a vitrificación, en una solución de 20% de Etilenglicol y un 20% de Dimetlsulfóxido, en los 2 soportes anteriormente descritos: Cryotop y asa de siembra. Por una parte los embriones serán desvitrificados y cultivados in vitro en una solución TCM199 con un 10% de suero a 38.5ºC y humedad saturada durante 48 horas. Se determinará la capacidad de desarrollo a blastocisto escapando o escapado. En un segundo experimento, embriones de ambos grupos experimentales serán transferidos mediante laparoscopia para evaluar la tasa de implantación a los catorce días tras la inseminación y el número de nacidos vivos a parto.[EN] Over the last several decades there have been many studies in order to develop efficient carriers that produce minimum damage over the cells. Cryotop is one of the more currently employed carriers for oocyte and embryo vitrification due to the high cooling rates achieved because it reduces the volume of vitrification, however, its price is around 21€ per each. The aim of this work is to compare between Cryotop and plastic inoculating loop for rabbit embryo vitrification. Briefly, 72 hours after insemination embryos were recovered in morulae or early blastocyst stage and were vitrified using vitrification solution consisting of 20% (vol/vol) ethylene glycol and 20% (vol/vol) dimethyl sulfoxide. Then embryos were loaded in Cryotop or inoculating loop. On the one hand, embryos were devitrified and cultured in vitro during 48 hours in medium TCM199 containing 10% serum at 38,5ºC and humidified atmosphere. It was evaluated the development of embryos until hatching. On the other hand, embryos from both experimental groups were transferred using the laparoscopic technique to evaluate implantation rate at fourteen days after insemination and offspring rate at birth. There were no differences between Inoculating loop and Cryotop under in vivo and in vitro culture conditions. Therefore, inoculating loop is a suitable method for replacing Cryotop.Almela Miralles, V. (2015). Desarrollo de un sistema económico para la vitrificación de embriones de conejo. http://hdl.handle.net/10251/54315.TFG

Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method

[EN] This study was designed to compare the efficiency of the Cryotop and Calibrated plastic inoculation loop (CPIL) devices for vitrification of rabbit embryos on in vitro development and implantation rate, offspring rate at birth and embryonic and fetal losses. CPIL is a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms. In experiment 1, embryos were vitrified using a Cryotop device and a CPIL device. There were no significant differences in hatched/hatching blastocyst stage rates after 48 h of culture among the vitrified groups (62±4.7% and 62±4.9%, respectively); however, the rates were significantly lower (P<0.05) than those of the fresh group (95±3.4%). In experiment 2, vitrified embryos were transferred using laparoscopic technique. The number of implanted embryos was estimated by laparoscopy as number of implantation sites at day 14 of gestation. At birth, total offspring were recorded. Embryonic and fetal losses were calculated as the difference between implanted embryos and embryos transferred and total born at birth and implanted embryos, respectively. The rate of implantation and development to term was similar between both vitrification devices (56±7.2% and 50±6.8% for implantation rate and 40±7.1% and 35±6.5% for offspring rate at birth); but significantly lower than in the fresh group (78±6.6% for implantation rate and 70±7.2% for offspring rate at birth, P<0.05). Likewise, embryonic losses were similar between both vitrification devices (44±7.2% and 50±6.8%), but significantly higher than in the fresh group (23±6.6%, P < 0.05). However, fetal losses were similar between groups (10±4.4%, 15±4.8% and 8±4.2%, for vitrified, Cryotop or CPIL and fresh, respectively). These results indicate that the CPIL device is as effective as the Cryotop device for vitrification of rabbit embryos, but at a cost of 0.05 per device.This research was supported by the projects Spanish Research project AGL2014-53405-C2-1-P Comision Interministerial de Ciencia y Tecnologia (FMJ, JSV) and Generalitat Valenciana research program (Prometeo II 2014/036, JSV, FMJ).Marco Jiménez, F.; Jiménez Trigos, ME.; Almela-Miralles, V.; Vicente Antón, JS. (2016). Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method. PLoS ONE. 11(2):1-9. https://doi.org/10.1371/journal.pone.0148661S1911

Image shows calibrated plastic inoculation loop and Cryotop devices.

<p>a) Both devices packaged in individual sterilized bag. b) Shows each device with the corresponding covers. c) Detail of the device covers.</p

Effect of vitrification device on implantation, offspring at birth and embryonic and fetal losses.

<p>Effect of vitrification device on implantation, offspring at birth and embryonic and fetal losses.</p

Effect of vitrification device after 48 h of in vitro culture.

<p>Effect of vitrification device after 48 h of in vitro culture.</p

Details of Calibrated plastic inoculation loop and Cryotop devices.

<p>a) Magnified image shows embryos in the corresponding devices. b) Shows each device with the corresponding covers (asterisks cotton top).</p