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

Equine Embryo TransferDOES THE EMBRYONIC CAPSULE IMPEDE THEFREEZING OF EQUINE EMBRYOS?E. Legrand, J. M. Krawiecki*, D. Tainturier † , P. Cornière, H. Delajarraud andJ. F. Bruyas †Progen, 50500 Carentan, France; *Veterinary Clinic, EAABC, 49400 Saumur, France; † Department <strong>of</strong>Reproduction, Nantes Veterinary School, BP 40706, 44307 Nantes, FranceThere has been no increase in <strong>the</strong> success rate <strong>of</strong>equine embryos freezing since 1982 (± 20 to 30%).A widely held view is that <strong>the</strong> younger (morula orearly blastocyst) (Slade et al. 1985; Skidmore et al.1991) and smaller (200 µm) (Slade et al. 1984;Lagneaux and Palmer 1991) <strong>the</strong> embryo is, <strong>the</strong>greater its chance <strong>of</strong> pregnancy after deep-freezingprocedure. Previous studies indicate that <strong>the</strong> capsuleplays a bigger part in <strong>the</strong> freezability <strong>of</strong> embryos inglycerol than <strong>the</strong> size or <strong>the</strong> stage (Legrand et al.1999; Bruyas et al. 2000). In order to verify this fact3 experiments have been conducted.EXPERIMENT 1The aim <strong>of</strong> <strong>the</strong> first experiment was to evaluate <strong>the</strong>relationship between <strong>the</strong> thickness <strong>of</strong> capsule and<strong>the</strong> cellular damage in embryos treated withcryoprotectant with or without freezing.Materials and methodsExamination <strong>of</strong> histological sections <strong>of</strong> 15embryos treated with glycerol and 15frozen/thawed embryos from previous studies(Bruyas et al. 1993; Bruyas et al. 1995; Legrand etal. 1999; Bruyas et al. 2000) was performed. Ineach <strong>of</strong> <strong>the</strong>se previous studies, each embryo, aftera classical procedure <strong>of</strong> glycerol incorporation andremoval and a classical freezing/thawing processusing glycerol as cryoprotectant, was incubatedduring 6 h at 37°C in order to underline cellulardamage. After culture, <strong>the</strong>y were fixed at 4°C inglutaraldehyde 2%, post fixed in 2% osmiumtetroxide, dehydrated in a graded ethanol seriesand embedded in epon 812. They were seriallysectioned into semi-thin sections (1 µm) and everyfifth section was stained with 0.5% hot toluidineblue for light microscopy.In <strong>the</strong> present study, nuclear and capsularstages were assessed using <strong>the</strong>se semi-thinsections. Each class <strong>of</strong> nuclei (pycnotic,caryorhexic and caryolytic for dead cells andinterphasic and mitotic for live cells) werecounted. Notation <strong>of</strong> <strong>the</strong> capsule thickness was asfollows: (Fig 1): 0 = no capsule; 1 = formingcapsule, a simple trace is detectable; 2 = sharp andthin capsule, sometime discontinued; 3 = sharpcapsule; 4 = thick capsule (0,8 µm). Four sections<strong>of</strong> each embryo were looked at, <strong>the</strong> capsular notewas determined as <strong>the</strong> mean <strong>of</strong> <strong>the</strong> 4 notes.Results and discussionEmbryos treated with glycerol without freezingand with a capsule graded from 0–3 showed anincrease (Fig 2) in <strong>the</strong> rate <strong>of</strong> dead cells, whereasthis rate decreased dramatically for embryos witha Grade 4 capsule (Fig 3).To explain that, our hypo<strong>the</strong>sis is as follow. Inembryos with a capsule noted 0–2, <strong>the</strong> water outflow and glycerol entry induce mild osmoticdamage. In embryos with a thicker capsule (Grade3) <strong>the</strong> water outflow cannot be compensated by <strong>the</strong>glycerol entry due to <strong>the</strong> thickness <strong>of</strong> <strong>the</strong> capsule.Therefore <strong>the</strong>re is a higher degree <strong>of</strong> osmoticdamage. Embryos with a thick capsule (Grade 4)presented <strong>the</strong> same morphology as fresh embryoswith a very low rate <strong>of</strong> dead cells. This observationsuggests that <strong>the</strong>re are no fluid movements through<strong>the</strong> capsule: no glycerol entry no water outflow,and <strong>the</strong>refore no osmotic damage.In <strong>the</strong> frozen/thawed groups (Fig 4), <strong>the</strong> rate <strong>of</strong>dead cells was directly proportional to <strong>the</strong> capsulethickness. This rate was respectively: 46.5 ±20.7% (Grade < 2), 57.4 ± 19.8 % (Grade 3), and85.4% ± 10.1 % (Grade 4). The embryos withthick capsule do not tolerate freezing-thawing62