A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species

SIMPLE SUMMARY: Commercially available devices with simultaneous vitrification of many embryos are scarce. In this study, we developed a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of embryos. The 3D technology was stereolithography, and the Cryoeyelet(®) device was printed in photosensitive resin. With the open Cryoeyelet(®), 25 late rabbit morulae/early blastocysts were vitrified per device and compared with the Cryotop(®) and the French mini-straw devices. Our results demonstrate that the CryoEyelet(®) device can be used for the vitrification of a high number of late morulae or early blastocyst rabbit embryos per device, yielding similar outcomes to the most used commercial devices based on minimum essential volume. ABSTRACT: Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet(®) device (n = 175; 25 embryos per device), the open Cryotop(®) device (n = 175; 10 embryos per device), and the traditional closed French mini-straw device (n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group (n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet(®) and the other devices. In experiment 2, the CryoEyelet(®) device showed a higher implantation rate compared with the Cryotop(®) (6.3% unit of SD, p = 0.87) and French mini-straw(®) (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet(®) device was similar to the Cryotop(®) device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet(®) showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes—a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet(®) device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet(®) device in other polytocous species for the simultaneous vitrification of a large number of embryos.