title: Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols.
abstract: OBJECTIVE
  To test the toxicity of cryoprotectant in sheep ovarian tissue and to determine optimal conditions for freezing hemiovary cortex.
  
  
  METHODS
  Small follicles (<60 microm in diameter) were isolated enzymatically for viability testing. Dead and live follicles were identified by using trypan blue staining, and follicle morphology was examined histologically.
  
  
  METHODS
  Centre hospitalo-universitaire de Biologie de la Reproduction, Hôpital Edouard Herriot, Lyon, France.
  
  
  METHODS
  Lambs 5 to 6 months of age.
  
  
  METHODS
  Two-millimeter slices of hemiovarian cortex were prepared for cryoprotectant toxicity tests and freezing procedures.
  
  
  METHODS
  Follicular mortality and histologic structure.
  
  
  RESULTS
  For freezing procedures, the concentration of cryoprotectant was increased to 2 M on the basis of results of cryoprotectant toxicity tests in fresh tissues. Follicular mortality rates were 4.6% with of 2 M dimethyl sulfoxide (DMSO) and 3.8% with 2 M of propylene glycol (PROH). After freezing with semiautomatic seeding, follicular mortality rates were 8.4% (2 M of DMSO) and 12.4% (2 M of PROH). Tissue morphology was well preserved with 1.5 M of DMSO or PROH. With 1.5 M DMSO, results of the slow cooling protocol (2 degrees C/min) without seeding and the standard very slow cooling protocol (0.3 degrees C/min) were similar.
  
  
  CONCLUSIONS
  Optimal survival of primordial follicles in the sheep was obtained by using a slow cooling protocol with semiautomatic seeding at 2 M of DMSO.
authors: Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon, Marcy l'Etoile, France. b.demirci@vet-lyon.fr
authors: B
authors: B
authors: Demirci
authors: J
authors: J
authors: Lornage
authors: B
authors: B
authors: Salle
authors: L
authors: L
authors: Frappart
authors: M
authors: M
authors: Franck
authors: J F
authors: JF
authors: Guerin
mesh_terms: D000818:Animals; D016923:Cell Death; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003470:Culture Media; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005260:Female; D009926:Organ Preservation / Q000379:methods*; D006080:Ovarian Follicle / Q000166:cytology* / Q000187:drug effects; D010053:Ovary / Q000166:cytology; D011409:Propylene Glycols / Q000494:pharmacology; D012756:Sheep; D013997:Time Factors
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media; D011409:Propylene Glycols; D004121:Dimethyl Sulfoxide