Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3TABLE 2: Str<strong>on</strong>g PAS-positive reacti<strong>on</strong>s foundin grafts recovered in Experiment 2Endometrium al<strong>on</strong>e- in mice with T elsewhere :0/7- in E+T sites :0/1Trophoblast al<strong>on</strong>e- in mice with E elsewhere :2/2- in E+T sites :2/4E+T :5/6Capsule :2/2TABLE 3: Mab OC-1 binding to grafts recoveredin Experiment 3Endometrium al<strong>on</strong>e- in mice with T elsewhere :0/2- in E+T sites :0/10Trophoblast al<strong>on</strong>e- in mice receiving no E :12/12- in mice with E elsewhere :2/2- in E+T sites :5/5E+T :10/104/6 sites c<strong>on</strong>taining T al<strong>on</strong>e (ei<str<strong>on</strong>g>the</str<strong>on</strong>g>r originally coengraftedwith E or with E at ano<str<strong>on</strong>g>the</str<strong>on</strong>g>r site in <str<strong>on</strong>g>the</str<strong>on</strong>g>same mouse), 5/6 successfully co-engrafted E+Tsites, and 0/8 grafts <str<strong>on</strong>g>of</str<strong>on</strong>g> E al<strong>on</strong>e (whe<str<strong>on</strong>g>the</str<strong>on</strong>g>roriginally co-engrafted with T or with T atano<str<strong>on</strong>g>the</str<strong>on</strong>g>r site in <str<strong>on</strong>g>the</str<strong>on</strong>g> mouse). There was nodiscernible increase <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> PAS-positive reacti<strong>on</strong>swith time (4–16 days).The PAS-positive material was more closelyrelated to T, or to <str<strong>on</strong>g>the</str<strong>on</strong>g> interface between T and E,than to E al<strong>on</strong>e. Weak PAS staining (a
Equine Embryo TransferDOES THE EMBRYONIC CAPSULE IMPEDE THEFREEZING OF <strong>EQUINE</strong> 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 <str<strong>on</strong>g>of</str<strong>on</strong>g>Reproducti<strong>on</strong>, Nantes Veterinary School, BP 40706, 44307 Nantes, FranceThere has been no increase in <str<strong>on</strong>g>the</str<strong>on</strong>g> success rate <str<strong>on</strong>g>of</str<strong>on</strong>g>equine embryos freezing since 1982 (± 20 to 30%).A widely held view is that <str<strong>on</strong>g>the</str<strong>on</strong>g> younger (morula orearly blastocyst) (Slade et al. 1985; Skidmore et al.1991) and smaller (200 µm) (Slade et al. 1984;Lagneaux and Palmer 1991) <str<strong>on</strong>g>the</str<strong>on</strong>g> embryo is, <str<strong>on</strong>g>the</str<strong>on</strong>g>greater its chance <str<strong>on</strong>g>of</str<strong>on</strong>g> pregnancy after deep-freezingprocedure. Previous studies indicate that <str<strong>on</strong>g>the</str<strong>on</strong>g> capsuleplays a bigger part in <str<strong>on</strong>g>the</str<strong>on</strong>g> freezability <str<strong>on</strong>g>of</str<strong>on</strong>g> embryos inglycerol than <str<strong>on</strong>g>the</str<strong>on</strong>g> size or <str<strong>on</strong>g>the</str<strong>on</strong>g> stage (Legrand et al.1999; Bruyas et al. 2000). In order to verify this fact3 experiments have been c<strong>on</strong>ducted.EXPERIMENT 1The aim <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> first experiment was to evaluate <str<strong>on</strong>g>the</str<strong>on</strong>g>relati<strong>on</strong>ship between <str<strong>on</strong>g>the</str<strong>on</strong>g> thickness <str<strong>on</strong>g>of</str<strong>on</strong>g> capsule and<str<strong>on</strong>g>the</str<strong>on</strong>g> cellular damage in embryos treated withcryoprotectant with or without freezing.Materials and methodsExaminati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> histological secti<strong>on</strong>s <str<strong>on</strong>g>of</str<strong>on</strong>g> 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 <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se previous studies, each embryo, aftera classical procedure <str<strong>on</strong>g>of</str<strong>on</strong>g> glycerol incorporati<strong>on</strong> andremoval and a classical freezing/thawing processusing glycerol as cryoprotectant, was incubatedduring 6 h at 37°C in order to underline cellulardamage. After culture, <str<strong>on</strong>g>the</str<strong>on</strong>g>y were fixed at 4°C inglutaraldehyde 2%, post fixed in 2% osmiumtetroxide, dehydrated in a graded ethanol seriesand embedded in ep<strong>on</strong> 812. They were seriallysecti<strong>on</strong>ed into semi-thin secti<strong>on</strong>s (1 µm) and everyfifth secti<strong>on</strong> was stained with 0.5% hot toluidineblue for light microscopy.In <str<strong>on</strong>g>the</str<strong>on</strong>g> present study, nuclear and capsularstages were assessed using <str<strong>on</strong>g>the</str<strong>on</strong>g>se semi-thinsecti<strong>on</strong>s. Each class <str<strong>on</strong>g>of</str<strong>on</strong>g> nuclei (pycnotic,caryorhexic and caryolytic for dead cells andinterphasic and mitotic for live cells) werecounted. Notati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> capsule thickness was asfollows: (Fig 1): 0 = no capsule; 1 = formingcapsule, a simple trace is detectable; 2 = sharp andthin capsule, sometime disc<strong>on</strong>tinued; 3 = sharpcapsule; 4 = thick capsule (0,8 µm). Four secti<strong>on</strong>s<str<strong>on</strong>g>of</str<strong>on</strong>g> each embryo were looked at, <str<strong>on</strong>g>the</str<strong>on</strong>g> capsular notewas determined as <str<strong>on</strong>g>the</str<strong>on</strong>g> mean <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> 4 notes.Results and discussi<strong>on</strong>Embryos treated with glycerol without freezingand with a capsule graded from 0–3 showed anincrease (Fig 2) in <str<strong>on</strong>g>the</str<strong>on</strong>g> rate <str<strong>on</strong>g>of</str<strong>on</strong>g> dead cells, whereasthis rate decreased dramatically for embryos witha Grade 4 capsule (Fig 3).To explain that, our hypo<str<strong>on</strong>g>the</str<strong>on</strong>g>sis is as follow. Inembryos with a capsule noted 0–2, <str<strong>on</strong>g>the</str<strong>on</strong>g> water outflow and glycerol entry induce mild osmoticdamage. In embryos with a thicker capsule (Grade3) <str<strong>on</strong>g>the</str<strong>on</strong>g> water outflow cannot be compensated by <str<strong>on</strong>g>the</str<strong>on</strong>g>glycerol entry due to <str<strong>on</strong>g>the</str<strong>on</strong>g> thickness <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g> capsule.Therefore <str<strong>on</strong>g>the</str<strong>on</strong>g>re is a higher degree <str<strong>on</strong>g>of</str<strong>on</strong>g> osmoticdamage. Embryos with a thick capsule (Grade 4)presented <str<strong>on</strong>g>the</str<strong>on</strong>g> same morphology as fresh embryoswith a very low rate <str<strong>on</strong>g>of</str<strong>on</strong>g> dead cells. This observati<strong>on</strong>suggests that <str<strong>on</strong>g>the</str<strong>on</strong>g>re are no fluid movements through<str<strong>on</strong>g>the</str<strong>on</strong>g> capsule: no glycerol entry no water outflow,and <str<strong>on</strong>g>the</str<strong>on</strong>g>refore no osmotic damage.In <str<strong>on</strong>g>the</str<strong>on</strong>g> frozen/thawed groups (Fig 4), <str<strong>on</strong>g>the</str<strong>on</strong>g> rate <str<strong>on</strong>g>of</str<strong>on</strong>g>dead cells was directly proporti<strong>on</strong>al to <str<strong>on</strong>g>the</str<strong>on</strong>g> 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
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