Follicular viability and morphology of sheep ovaries after exposure to cryoprotectant and cryopreservation with different freezing protocols.
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.
Demirci
B
B
Laboratoire de Zootechnie, Ecole Nationale Vétérinaire de Lyon, Marcy l'Etoile, France. b.demirci@vet-lyon.fr
Lornage
J
J
Salle
B
B
Frappart
L
L
Franck
M
M
Guerin
J F
JF
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
D003451:Cryoprotective Agents; D003470:Culture Media; D011409:Propylene Glycols; D004121:Dimethyl Sulfoxide