Proceedings of the 5th International Symposium on EQUINE ...

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Havemeyer Foundation Monograph Series No. 3Capsule note 0Capsule note 1pellucidpellucidcapsuleinterphasicnucleusinterphasicnucleusnucleolusExternalmediumCapsule note 2Capsule note 3pellucidpellucidcapsulecapsuletrophoblasticcellsCapsule note 4Capsule note 4pellucidpellucidcapsulenecrotic cellscapsuletrophoblasticcellsembryoniccellsblastocoeleFig 1: Embryonic capsule notation.process, and <strong>the</strong> embryos without capsule are lessdamaged than o<strong>the</strong>rs.The explanation could be that embryoniccapsule interferes with <strong>the</strong> cryoprotective effectsby preventing glycerol entry in embryonic cells.EXPERIMENT 2In order to remove this difficulty <strong>the</strong> purpose <strong>of</strong> <strong>the</strong>second experiment was to test <strong>the</strong> effects <strong>of</strong>enzymatic treatments on <strong>the</strong> capsule so as to deleteor to permeabilise it.Materials and methodsThe effect on <strong>the</strong> embryo was also observed.Two enzymes, trypsin (EC 3.4.21.4) andcollagenase (EC 3.4.24.3), have been used indifferent concentrations (Trypsin: 0.005%,0.01%, 0.025%, 0.05%, 0.1%, 0.25%, 0.5%, 1%,63
Equine Embryo TransferNecrotic rate (%)807060504030201000 1 2 3 4Capsular noteFig 2. Relation between necrotic rate and capsularthickness in embryos without freezing procedure.Necrotic rate10090807060504030201000 1 2 3 4Capsule noteBruyas et al. 1993 (glycerol 1.4M)Legrand et al. 1999 (glycerol 1M)Bruyas et al. 2000 (glycerol 1.5M)Fig 4. Relationship between necrotic rate and capsularthickness in frozen/thawed embryos.Necrotic rate (%)7060504030201000 1 2 3 4Necrotic rate (%)10090807060504030201000.005 0.01 0.025 0.05 0.1 0.5 1 5Concentration (%)Fig 3. Model graph <strong>of</strong> <strong>the</strong> relationship between necroticrate and capsular importance.Fig 5: Effect <strong>of</strong> trypsin treatment on embryos treatedwith glycerol without freezing/thawing process.TABLE 1: pregnancy rate according to <strong>the</strong> age and size <strong>of</strong> embryosPregnancy Age <strong>of</strong> embryos (days post ovulation) Diameter <strong>of</strong> embryos (µm)diagnoses 5.5 6.5 7.5 8.5 < 200 200–500 500–1000 > 1000Day 14 0/1 1/1 2/2 3/4 2/3 1/1 0/1 3/3Day 21 0/1 1/1 0/1* 2/4 1/3 0/0* 0/1 2/3Day 280/1 1/1 0/1* 2/4 1/3 0/0* 0/1 2/3* a pregnant mare was injured between Day 14 and Day 215% and collagenase : 0.5%, 1%, 2.5%, 5%).Thirteen Day 8 embryos were placed in PBSmedium + enzyme for 15 min, <strong>the</strong>n glycerol wasadded in 3 steps (0.5 M for 10 min; 1 M for 10min; 1.5 M for 10 min) and immediatelyremoved in 3 steps (Glycerol (G)1.25 M +sucrose (s) 0.25 M for 10 min; G 0.6 M + s 0.25M for 10 min; s 0.25 M for 10 min) withoutfreezing. Histological analysis was performed asin <strong>the</strong> first experiment.Results and discussionNecrotic rate with trypsin treatment is reported inFigure 5.Data show that: i) trypsin seemed to be betterto increase <strong>the</strong> permeability <strong>of</strong> <strong>the</strong> capsule thancollagenase; and ii) low enzymatic concentrations(1%) were toxic for embryoniccells.64
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