title: Cellular damage suffered by equine embryos after exposure to cryoprotectants or cryopreservation by slow-freezing or vitrification.
abstract: BACKGROUND
  Equine embryos are cryopreserved by slow-freezing or vitrification. While small embryos (<300 μm) survive cryopreservation reasonably well, larger embryos do not. It is not clear if slow-freezing or vitrification is less damaging to horse embryos.
  
  
  OBJECTIVE
  To compare the type and extent of cellular damage suffered by small and large embryos during cryopreservation by slow-freezing vs. vitrification.
  
  
  METHODS
  Sixty-three Day 6.5-7 embryos were subdivided by size and assigned to one of 5 treatments: control, exposure to slow-freezing or vitrification cryoprotectants (CPs), and cryopreservation by either technique.
  
  
  METHODS
  After thawing/CP removal, embryos were stained with fluorescent stains for various parameters of cellular integrity, and assessed by multiphoton microscopy.
  
  
  RESULTS
  Exposing large embryos to vitrification CPs resulted in more dead cells (6.8 ± 1.3%: 95% confidence interval [CI], 3.1-10.4%) than exposure to slow-freezing media (0.3 ± 0.1%; 95% CI 0.0-0.6%: P = 0.001). Cryopreservation by either technique induced cell death and cytoskeleton disruption. Vitrification of small embryos resulted in a higher proportion of cells with fragmented or condensed (apoptotic) nuclei (P = 0.002) than slow-freezing (6.7 ± 1.5%, 95% CI 3.0-10.4% vs. 5.0 ± 2.1%, 95% CI 4.0-14.0%). Slow-freezing resulted in a higher incidence of disintegrated embryos (P = 0.01) than vitrification. Mitochondrial activity was low in control embryos, and was not differentially affected by cryopreservation technique, whereas vitrification changed mitochondrial distribution from a homogenous crystalline pattern in control embryos to a heterogeneous granulated distribution in vitrified embryos (P = 0.05).
  
  
  CONCLUSIONS
  Cryopreservation caused more cellular damage to large embryos than smaller ones. While vitrification is more practical, it is not advisable for large embryos due to a higher incidence of dead cells. The choice is less obvious for small embryos, as vitrification led to occasionally very high percentages of dead or damaged cells, but a lower incidence of embryo disintegration. Modifications that reduce the level of cellular damage induced by vitrification are required before it can be considered the method of choice for cryopreserving equine embryos.
authors.affiliation: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: W K
authors.initials: WK
authors.lastname: Hendriks
authors.affiliation: Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: B A J
authors.initials: BA
authors.lastname: Roelen
authors.affiliation: Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: B
authors.initials: B
authors.lastname: Colenbrander
authors.affiliation: Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, The Netherlands.
authors.forename: T A E
authors.initials: TA
authors.lastname: Stout
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000009:adverse effects*; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000187:drug effects*; D047108:Embryonic Development; D005615:Freezing; D006736:Horses / Q000196:embryology*; D013997:Time Factors; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents
keywords: cryopreservation; embryo; horse; slow-freezing; vitrification