Development of Cheaper Embryo Vitrification Device Using the Minimum Volume Method.

Francisco Marco-Jiménez,Estrella Jiménez-Trigos,Victoria Almela-Miralles,José Salvador Vicente,Yann Herault

doi:10.1371/journal.pone.0148661

Francisco Marco-Jiménez, Estrella Jiménez-Trigos + Show 3 more

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https://doi.org/10.1371/journal.pone.0148661

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Abstract

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.

Highlights

Vitrification was introduced in 1985 as a simple and cheap way to cryopreserve mammalian embryos in the absence of ice [1]
We propose the use of disposable, sterile calibrated plastic inoculation loop (CPIL), a simple tool used mainly by microbiologists to retrieve an inoculum from a culture of microorganisms
The rate of implantation and development to term was significantly lower in the vitrified groups than in the fresh control (56±7.2% and 50±6.8% vs 78±6.6% for implantation rate and 40±7.1% and 35±6.5% vs 70±7.2% for offspring rate at birth, for vitrified, Cryotop or Calibrated plastic inoculation loop (CPIL) vs fresh, respectively, P < 0.05, Table 2), with no differences between the groups vitrified

Summary

Introduction

Vitrification was introduced in 1985 as a simple and cheap way to cryopreserve mammalian embryos in the absence of ice [1]. The first successful vitrification was done with mouse embryos using a relatively large volume sample (0.25-mL straw). Arav [2,3,4,5,6] introduced the idea of using the same technique for vitrification in a small drop, which he later designated the “minimum drop size”. Vitrification of embryos, on the other hand, initially attempted in the late 1980s, was not applied clinically until recently [7]. Vitrification is being widely utilized in livestock and human embryos [8]

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