Cryopreservation of equine oocytes: looking into the crystal ball

Abstract

In vitro embryo production has evolved rapidly in the horse over the past decade, but blastocyst rates from vitrified equine oocytes remain quite poor and further research is needed to warrant application. Oocyte vitrification is affected by several technical and biological factors. In the horse, short exposure of immature oocytes to the combination of permeating and non-permeating cryoprotective agents has been associated with the best results so far. High cooling and warming rates are also crucial and can be obtained by using minimal volumes and open cryodevices. Vitrification of in vivo-matured oocytes has yielded better results, but is less practical. The presence of the corona radiata seems to partially protect those factors that are necessary for the construction of the normal spindle and for chromosome alignment, but multiple layers of cumulus cells may impair permeation of cryoprotective agents. In addition to the spindle, the oolemma and mitochondria are also particularly sensitive to vitrification damage, which should be minimised in future vitrification procedures. This review presents promising protocols and novel strategies in equine oocyte vitrification, with a focus on blastocyst development and foal production as most reliable outcome parameters.

New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification

“…Gamete cryopreservation would further increase ICSI flexibility by allowing to postpone the decision on the fertilizing stallion. Moreover, oocytes could be salvaged from recently deceased animals in places where ICSI is not available; oocytes might be collected and stored outside the reproductive season, and good-quality germ lines of young mares could be preserved before they are enrolled in competition [ 5 , 6 ]. Finally, horse oocyte banks would allow the worldwide spread of valuable female genetics for commercial purposes, for breeding programs of endangered equid breeds and species, or for research in any ART in horses [ 5 , 7 ].…”

“…Moreover, oocytes could be salvaged from recently deceased animals in places where ICSI is not available; oocytes might be collected and stored outside the reproductive season, and good-quality germ lines of young mares could be preserved before they are enrolled in competition [ 5 , 6 ]. Finally, horse oocyte banks would allow the worldwide spread of valuable female genetics for commercial purposes, for breeding programs of endangered equid breeds and species, or for research in any ART in horses [ 5 , 7 ]. However, the mammalian oocyte is one of the hardest cells to cryopreserve [ 8 ], and, up to now, the efficiency of equine oocyte cryopreservation is limited.…”

“…The vitrification of immature and in vitro matured equine oocytes compromises embryo development after ICSI [ 11 , 12 , 13 , 14 ]. Hence, in order to exploit the application potential of equine oocyte vitrification, further optimization of the vitrification protocols is necessary (for review see De Coster & Angel-Velez, et al, 2020 [ 5 ]).…”

“…In humans and mice, the oocyte vitrification in a trehalose-containing medium was also associated with higher blastocyst rates than vitrification with sucrose [ 31 ], but the differences were not always significant [ 32 , 33 ], probably due to variations in the protocols and concentrations. Besides these disaccharides, which have been the only non-permeating CPAs tested in equine oocytes vitrification up to now (for review, De Coster & Angel-Velez, et al, 2020 [ 5 ]), monosaccharides may provide a valuable alternative. Monosaccharides (galactose, fructose, glucose) seem to be more effective osmotic buffers than disaccharides during vitrification and warming [ 34 ], and they have been used successfully for the vitrification of bovine [ 35 ] and feline [ 36 ] oocytes, as well as for bovine [ 37 ], porcine [ 38 ], dromedary camel [ 39 ], alpaca [ 40 ], and equine embryos [ 37 , 41 , 42 ].…”

“…A superior standard protocol describing all the aspects of oocyte cryopreservation has not yet been identified in the horse. Multiple factors influence the success of equine oocyte vitrification, and one crucial technical aspect is the type of CPA [ 5 ]. Therefore, the aim of the present study was to determine the optimal combination of CPAs for the vitrification of immature equine oocytes.…”