An Update on Equine Embryo Cryopreservation: A Glass Act where Size matters

Historically the cryopreservation of equine embryos has been problematic. In all species, smaller embryos with compact cells or small blastocoele cavities tend to survive cryopreservation better than larger embryos. Obtaining such embryos from the horse is difficult. The equine embryo does not enter the uterus until 5.5–6 days post ovulation after which it rapidly expands forming a large blastocoele cavity. In addition, around day 7 the equine embryo becomes enveloped by a glycoprotein coating, the equine blastocyst capsule, which is believed to inhibit the passage of cryoprotectants into the embryo. Research into embryo cryopreservation has also been hampered due to our inability to superovulate mares, meaning obtaining sufficient embryos for experiments is costly and time-consuming. Despite these problems, progress has been made and, if one is judicious in the timing of embryo collection and cryopreservation techniques, it is now possible to obtain high pregnancy rates following the transfer of thawed-frozen embryos.

Two techniques are available to cryopreserve equine embryos: slow freezing and vitrification. Both techniques rely on cryoprotectant agents (CPAs) to protect the embryo from cellular damage during the freezing process. Slow freezing typically uses low concentrations of CPAs and embryos are frozen in straws with a slow controlled rate of freezing. In contrast, vitrification uses higher concentrations of CPAs and rapid cooling rates to transform the vitrification solution in and around the cells into a glassy state without ice crystal formation. Effective vitrification requires embryos to be placed in minimal volumes of vitrification medium on low-volume holders before being plunged into liquid nitrogen.

Most in vivo derived embryos are currently cryopreserved using vitrification techniques, which this presentation will concentrate on. Colorado State University led the way forward for commercial vitrification by demonstrating acceptable pregnancy rates (62%; 16/26) following exposure of embryos to a 3-step ethylene glycol and glycerol protocol [1]. This research translated into a commercial vitrification kit although, to achieve success, embryos needed to be ≤250 μm in diameter and the zona pellucida no less than half its normal thickness. Although this is challenging under clinical conditions, pregnancy rates using this methodology have been reported at around 60% (59%, 38/64)[2].

Despite the success with smaller embryos, pregnancy rates following vitrification of larger embryos remained depressingly low. However, real progress was reported by Choi et al. [3] when it was discovered that micromanipulation-assisted puncture of the capsule and collapse of the blastocoele cavity while undertaking trophoblast biopsies was compatible with embryo survival following their subsequent transfer to recipient mares. This led the authors to vitrify embryos (407–565 μm) following puncture and aspiration of ≥95% of their blastocoele fluid and they achieved a pleasing 71% (5/7) pregnancy rate following their subsequent warming and transfer to recipient mares [4]. Other groups showed similarly high pregnancy rates using puncture and aspiration techniques prior to vitrification with subsequent warming and transfer to recipient mares [5,6]. Vitrification protocols varied between these studies but all achieved their best results using minimal volume devices, rather than 0.25 mL straws, to load the embryo on prior to plunging into liquid nitrogen. Furthermore, it was deemed that puncture of the blastocyst capsule and removal of blastocoele fluid prior to vitrification was essential for post-warming embryo survival.

More recent studies have demonstrated that, although puncture of the capsule is required for successful vitrification, aspiration of the blastocoele fluid is not essential for embryos ≤500 μm with comparable pregnancy rates regardless of aspiration or not (aspirated 80% [8/10] vs. nonaspirated 75% [9/12]; P = 0.816), whereas there was a clear advantage of aspiration in embryos >550 μm (aspirated 72% [13/18] vs. nonaspirated 10% [1/10]; P = 0.006) [7]. Since aspiration of embryos requires the expense of a micromanipulator mounted on an inverted microscope coupled with user-skill, the discovery that smaller embryos did not require aspiration led to a manual technique being developed for embryos <560 μm. The use of an ultrafine, tungsten handheld needle (tip 1 μm) combined with a steady hand can provide an effective way to puncture embryos with a reported pregnancy rate of 82% (14/17) for embryos ≤560 μm treated in this manner [8]. Larger (>560 μm) manually punctured embryos failed to give acceptable pregnancy rates (10%; 1/10), demonstrating, once again, that larger embryos require aspiration of the blastocoele fluid prior to vitrification. This study also showed that neither puncture nor aspiration was required for embryos ≤300 μm, regardless of zona thickness, to survive vitrification and form pregnancies when warmed and transferred (80%; 8/10).

Hence, embryos ≤300 μm can be vitrified without puncture or aspiration, whereas embryos >300 μm but ≤550 μm require puncture but not necessarily aspiration prior to vitrification. With a steady hand and a little practice, puncture of such embryos can be accomplished manually. Embryos >550 μm require puncture and aspiration of the blastocoele fluid, presently only achievable using a micromanipulator. For those without a micromanipulator, and from a practical point of view, flushing mares early on day 7 post ovulation helps to ensure embryos ≤550 μm are more likely to be recovered. Even when using a micromanipulator to achieve puncture and aspiration, embryos >800 μm tend to have lower pregnancy rates following warming and transfer. Optimising protocols may help to overcome this, although these larger embryos may be more sensitive to vitrification per se due to developmental changes that occur around this time, such as the development of the endoderm.

In conclusion, we have come a long way since the first thawed-frozen embryo made it to term following its transfer in the early 1980s [9]. Forty years on and we now have vitrification protocols that can achieve high pregnancy rates for most embryos. Making such protocols increasingly user-friendly should help further expand the use of cryopreservation.

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