Santos R R, Tharasanit T, Figueiredo J R, van Haeften T, van den Hurk R
Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands.
Cell Tissue Res. 2006 Sep;325(3):523-31. doi: 10.1007/s00441-006-0193-5. Epub 2006 Apr 28.
Caprine preantral follicles within ovarian fragments were cryopreserved in the absence or presence of 0.5 M sucrose with or without 1 M dimethyl sulfoxide and/or 1 M ethylene glycol (EG). After being thawed, they were washed in minimum essential medium with or without 0.3 M sucrose. Histological analysis of follicle integrity immediately after cryopreservation showed consistent beneficial effects of including sucrose in the three cryoprotectant solutions analyzed when tissue was thawed without sucrose (53.9+/-14.8-82.4+/-3.2% normal vs 27.6+/-1.6-36.6+/-6.5%, P<0.05). However, in further studies, the addition of sucrose to the thaw solutions proved detrimental or of no benefit. An analysis of the cryopreserved material with calcein-AM and ethidium homodimer (markers for living and dead cells, respectively) gave comparable results to those obtained by histology. Follicles cryopreserved in EG, EG plus sucrose, or sucrose alone were cultured in vitro for 24 h following warming. During this culture period, viability fell most rapidly in material cryopreserved in sucrose alone and was no longer correlated with either the viability or integrity estimates made immediately after warming. By contrast, the viability of follicles cryopreserved in EG with sucrose and then cultured for 24 h was not significantly different from the cultured non-frozen controls. These results indicate that cryopreservation in 1 M EG plus 0.5 M sucrose combined with thawing without sucrose is effective for caprine ovarian tissue.
将卵巢组织碎片中的山羊窦前卵泡在有或无0.5M蔗糖的情况下进行冷冻保存，同时添加或不添加1M二甲基亚砜和/或1M乙二醇（EG）。解冻后，将它们在有或无0.3M蔗糖的最低限度基本培养基中洗涤。冷冻保存后立即对卵泡完整性进行组织学分析表明，当组织在无蔗糖的情况下解冻时，在所分析的三种冷冻保护剂溶液中加入蔗糖具有一致的有益效果（正常卵泡比例为53.9±14.8 - 82.4±3.2%，而未添加蔗糖时为27.6±1.6 - 36.6±6.5%，P<0.05）。然而，在进一步研究中，向解冻溶液中添加蔗糖被证明是有害的或没有益处。用钙黄绿素 - AM和溴化乙锭同二聚体（分别为活细胞和死细胞的标记物）对冷冻保存材料进行分析，得到了与组织学分析相当的结果。在解冻后，将在EG、EG加蔗糖或仅蔗糖中冷冻保存的卵泡体外培养24小时。在此培养期间，仅在蔗糖中冷冻保存的材料活力下降最快，并且不再与解冻后立即进行的活力或完整性评估相关。相比之下，在EG加蔗糖中冷冻保存然后培养24小时的卵泡活力与未冷冻的培养对照没有显著差异。这些结果表明，1M EG加0.5M蔗糖的冷冻保存方法并结合无蔗糖解冻对山羊卵巢组织是有效的。
