Crypreservation of equine embryos 2/5/18

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1 Introduction Cryopreservation of equine embryos Carolina Herrera, M Sc. Embryo cryopreservation has been successfully used in human and bovine embryos for more than twenty years. Clinic for Reproductive Medicine, Vetsuisse - Faculty, University of Zurich Embryo Cryopreservation Slade et al Lascombes and Pashen 2000 Eldridge-Panuska WD et al Choi et al Diaz et al Sanchez et al < 300 um um um um 1 2 Introduction Human embryos Equine large embryo cryopreservation (>300 µm) Mayor difficulties Large size of embryos (200 to 1200 um in diameter) 140 μm 200 μm Bovine embryos 160 μm 230 μm Relative size of embryos Capsule impermeable to cryoprotectants Equine embryos Overcome difficulties : collect embryos on day 6 Lower recovery rates!! μm 300 μm 600 μm 900 μm 1200 μm 1500 μm 1800 μm Choi et al. Commercial vitrification kits Successful cryopreservation of expanded equine blastocysts Flushing Embryo biopsy Large equine embryos (>300 um) need to be collapsed before cryopreservation Agtech Equine Vitrification Kit Bioniche, Syngro Equine Vitrification Kit Preimplantation Genetic Diagnosis (PGD) Embryo Cryopreservation 5 6 Switzerland 1

Crypreservation of equine embryos 2/5/18 Materials and Methods 570 in vivo produced

much blastocoele fluid as possible 33% 58% < 300 um 300-750 um > 750 um Immediately

Vitrification Solution 2 (VS2) and load in hemistraw within 45 Plunge in liquid

2 Crypreservation of equine embryos 2/5/18 Materials and Methods 570 in vivo produced equine embryos 9% Using a microscope + micromanipulator collapse embryo by removing as much blastocoele fluid as possible 33% 58% < 300 um um > 750 um Immediately start vitrification process: Vitrification Solution 1 (VS1) for 3 to 5 minutes Vitrification Solution 2 (VS2) and load in hemistraw within 45 Plunge in liquid nitrogen Seal, label and store Store blastocoele fluid for PCR Materials and Methods Switzerland 2

3 Stereomicroscope Small volume (<10µl) pipette and sterile tips Hemistraw Vitrification solutions Liquid nitrogen Trained operator!!! New low cost and portable version Stereomicroscope Small volume (<10µl) pipette and sterile tips Switzerland 3

Hemistraw Vitrification solutions Vitrification solution

5% (v/v) dimethylsulfoxide Vitrification solution (VS2)

5 M sucrose 19 20 Liquid nitrogen Previous experience in

with experienced professional Trained operator!

vitrified embryo 750 um Fresh embryo Post warming 72

4 Hemistraw Vitrification solutions Vitrification solution (VS1) (Vajta et al.) 7.5% (v/v) ethylene glycol 7.5% (v/v) dimethylsulfoxide Vitrification solution (VS2) (Vajta et al) 16.5% (v/v) EG 16.5% (v/v) DMSO 0.5 M sucrose Liquid nitrogen Previous experience in vitrification If no experience, plan proper training with experienced professional Trained operator!!! Collapsed vitrified embryo 1000 um Collapsed vitrified embryo 750 um Fresh embryo Post warming 72 hours IVC (650 um) Fresh embryo Post warming 24 hours IVC (1100 um) Switzerland 4

Embryos can be shipped to a different location for cryopreservation.

5 Clinical application of embryo cryopreservation Summary Cryopreservation of equine embryos is now possible, although this technique is not widely used in commercial programs. The actual technique requires expensive equipment and trained personnel. Embryos can be shipped to a different location for cryopreservation. Setting 1 Setting 2 Setting Clinic of Reproductive Medicine Vetsuisse Faculty, University of Zrich Sarah Wyck Elise Jeannerat Andreas Fleisch Fredi Janett Stefan Bauersachs Heinrich Bollwein THANK YOU Switzerland 5

Protocol for embryo vitrification using open pulled straws. Introduction. Reagents. Materials

Protocol for embryo vitrification using open pulled straws Froylan Sosa and Peter J Hansen Department of Animal Sciences, University of Florida Introduction This vitrification protocol is a slight modification