Positive impact of sucrose supplementation during slow freezing of cat ovarian tissues on cellular viability, follicle morphology, and DNA integrity.
The objectives of the study were to (1) examine and optimize the impact of sucrose during slow freezing and (2) compare the results of two freezing methods (slow freezing and vitrification) on cellular viability (germinal and stromal cells), follicle morphology, DNA integrity, and gap junction protein expression (connexin 43 [Cx 43]). Different sucrose supplementations (0, 0.1, and 0.3 M) in standard freezing medium were compared before and after slow freezing. Ovarian tissue slow frozen using 0.1- (4.0 ± 0.4) or 0.3-M sucrose (3.9 ± 0.5) yielded better follicular viability (number of positive follicles per 0.0625 mm(2)) than the group without sucrose (1.9 ± 0.2; P < 0.05). Morphologically normal primordial follicles were higher in the sucrose-treated groups (0.1 M, 47.4% and 0.3 M, 43.5%) than the group without sucrose (0 M, 33.8%; P < 0.05). Moreover, less apoptotic primordial follicles were found in both sucrose groups (0.1 M, 1.2% and 0.3 M, 1.9%) than the group without sucrose (7.7%; P < 0.05). However, their Cx 43 expression showed no difference among the groups of different sucrose concentrations. In terms of the freezing methods used, vitrified ovarian tissues had fewer viable follicles (3.2 ± 0.6) than the slow-freezing method (4.6 ± 0.6; P < 0.05). In addition, the slow freezing resulted in more postthawed morphologically normal primordial follicles (38.8% vs. 28.3%, P < 0.05) and less apoptotic primordial follicles (3.8% vs. 8.9%, P < 0.05) than vitrification. The Cx 43 expression showed no difference between slow freezing and vitrification. The present study reported the positive effects of sucrose supplementation and slow-freezing method on the follicular viability, follicular histologic appearances of follicles, and apoptosis of the follicles and stromal cells in cat ovarian tissues.
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Nae
N
Tanpradit
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Washington, DC, USA.
Pierre
P
Comizzoli
Department of Anatomy, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Sayamon
S
Srisuwatanasagul
Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. Electronic address: kaywalee.c@chula.ac.th.
Kaywalee
K
Chatdarong
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002415:Cats; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D004249:DNA Damage; D005260:Female; D006080:Ovarian Follicle / Q000033:anatomy & histology / Q000187:drug effects / Q000502:physiology*; D010053:Ovary / Q000502:physiology*; D013395:Sucrose / Q000494:pharmacology*; D058989:Vitrification
D013395:Sucrose
Connexin 43; Cryopreservation; Domestic cat; Ovarian tissue; Sucrose; Vitrification