Successful vitrification of manually punctured equine embryos

Abstract

Background: Successful vitrification of equine expanded blastocysts requires collapse of the blastocoele cavity using a micromanipulator-mounted biopsy pipette on an inverted microscope. Such equipment is expensive and requires user skill.Objectives: To develop a manual method of blastocoele collapse prior to vitrification using commercial products. Study design: In vivo experiment.Methods: Seventy-nine Day 7 or 8 embryos were measured and graded. Twenty were vitrified following micromanipulator-assisted puncture and aspiration before being used to validate commercial human vitrification and warming kits containing, respectively, 2-step concentrations of DMSO and ethylene glycol (7.5%-15% v:v) and decreasing concentrations of sucrose. After warming, embryos were transferred to recipient mares. Once validated, the commercial kits were used to vitrify and warm a further 39 embryos which were punctured manually using a microneedle, 2 (5%) were damaged during puncture and excluded; 20 more embryos were vitrified without puncture. Embryos were grouped as follows: non-punctured ≤ 300µm (n = 10) and >300 to ≤560 µm (n = 10), punctured small (>300 to ≤560 µm; n = 17) and large (>560 µm; n = 10) and exposed to the equilibration solution (ES) in the kit for 6min.An additional group of punctured large embryos was exposed to ES for 8min (n = 10).For the initial warming step, embryos were exposed for 1min to the thawing solution at 42°C, before being moved to a dilution solution at room temperature.Results: Vitrified, manually punctured embryos ≤560 µm exposed to ES for 6min resulted in a pregnancy rate of 82% (14/17). Unpunctured embryos ≤300 µm gave an 80% (8/10) pregnancy rate. Larger unpunctured embryos, punctured embryos >560 µm and embryos exposed to ES for 8min gave significantly reduced pregnancy rates.Main limitations: Limited group sizes. Conclusion:High pregnancy rates can be achieved by manually puncturing ≤560 µm equine embryos prior to their vitrification and subsequent warming in commercial media.

The Mare: A Pertinent Model for Human Assisted Reproductive Technologies?

“…However, this method requires a micromanipulator, which is too expensive and too complicated to become widespread in the equine embryo transfer industry. Very recently good pregnancy rates were obtained with >300 µm embryos that were vitrified after manual blastocoel puncture [ 187 ]. If this is confirmed, this technique can be used on a larger scale and, unlike humans and other ruminants, the freezing of equine embryos may develop rapidly.…”

WR 'Twink' Allen: A career revolutionising the study and practice of equine reproduction

“…14 Subsequently, the conundrum of how to reliably cryopreserve the larger horse blastocysts recovered by flushing on day 7 or 8 was solved by the discovery that the key was reducing the blastocoele fluid volume by puncture and aspiration; indeed, recent studies by Twink and Sandra Wilsher have helped better define the relationship between the diameter of expanded horse blastocysts and the manipulations required to enable successful vitrification. 32,33 Cryopreservation was not the only reason Twink and colleagues wanted to work with very early embryos. Obtaining early embryos, when all or most of the cells were still pluripotent, was essential for the production of genetically identical monozygotic twins.…”

Colección, vitrificación y transferencia post-calentamiento de embriones equinos producidos in vivo: una revisión de literatura

“…If small, they do not require capsule perforation (Panzani et al, 2012a;Fanelli et al, 2020). However, if large they might preferentially be punctured before the vitrification step (Hochi et al, 1995;Eldridge-Panuska et al, 2005;Scherzer et al, 2011;Wilsher et al, 2019;Fanelli et al, 2020;Wilsher et al, 2021). In general, the post-ovulation day selected to attempt an embryo recovery influences the percent of embryos collected.…”

“…30 uL (Eldridge-Panuska et al, 2005;Fanelli et al, 2020). On the other hand, if the equine embryos are large, besides the capsule perforation (Wilsher et al, 2021) and blastocoelic fluid extraction (Sanchez et al, 2017;Herrera, 2021) they will preferentially require direct contact with liquid nitrogen (Díaz et al, 2016;Sanchez et al, 2017;Herrera, 2021). It seems that with large equine embryos (besides capsule perforation and blastocoelic fluid extraction), when comparing between open and closed vitrification embryo carriers which allow direct and indirect contact with liquid nitrogen during the vitrification step; that the open systems promotes a higher TTI and hence augments the PVSPR (Sanchez et al, 2017).…”

“…Furthermore, the volume of liquid used to hold large embryos during the vitrification step might be preferentially small (usually < 1 uL) to increase the TTI (Díaz et al, 2016;Sanchez et al, 2017). Another factor that might influence embryo survival is the diameter of the micropipette employed to perforate its capsule; however, limited and not conclusive data addressing this topic exist (Choi et al, 2010;Díaz et al, 2016;Ferris et al, 2016;Sanchez et al, 2017;Wilsher et al, 2021). Finally, the liquid solutions selected to transfer embryos into the uterus of mares or jennies might be carefully analyzed.…”