Lopes Kátia Regina F, Praxedes Erica Camila G, Campos Livia B, Bezerra Marcelo B, Lima Gabriela L, Saraiva Márcia Viviane A, Silva Alexandre R
Laboratory of Animal Germplasm Conservation-LCGA, Universidade Federal Rural do Semi-Arido-UFERSA, Mossoró, RN, Brazil.
Reprod Domest Anim. 2018 Oct;53(5):1060-1067. doi: 10.1111/rda.13203. Epub 2018 May 29.
The aim of this study was to assess a vitrification protocol for asinine ovarian tissue, to preserve preantral follicles using different cryoprotectant solutions, composed of various concentrations (EG 3 M or 6 M) of dimethyl sulfoxide or ethylene glycol isolate, or as a combination (DMSO 3 M + EG 3 M). Ten pairs of ovaries from Brazilian north-eastern breed jennies were obtained through videolaparoscopy, and cortical fragments were submitted to a solid-surface vitrification (SSV) using each cryoprotectant solution. The ovarian tissue was evaluated for follicular morphology and viability, DNA integrity (TUNEL technique) and the presence of nucleolar organizing regions in granulosa cells (AgNOR technique). After thawing, the percentage of normal preantral follicles was significantly reduced in the vitrified ovarian tissue fragments compared to the fresh control (p < 0.05). When comparing treatments, the use of DMSO 3 M (81.7 ± 37.5%), EG 3 M (83.7 ± 27.4%) and the combination of both DMSO 3 M + EG 3 M (81.8 ± 46.8%) allowed a greater percentage of follicular survival in contrast to DMSO 6 M (69.8 ± 16.5%) and EG 6 M (72.3 ± 18.0%; p < 0.05). When vitrified using the DMSO + EG combination, a higher percentage (62.5 ± 29.1%) of viable follicles (trypan blue) was observed in relation to the other vitrification treatments (p < 0.05). The TUNEL technique identified that all treatments tested showed DNA fragmentation in the follicular cells, except in the case of the DMSO 3 M + EG 3 M treatment. When evaluating the presence of NORs, no significant differences were observed in the amount of NORs between the fresh and vitrified groups using DMSO 3 M + EG 3 M (p > 0.05). We concluded that the combination DMSO 3 M + EG was more efficient for the vitrification of ovarian tissue taken from Equus asinus, allowing adequate preservation of PAFs morphology, viability, DNA integrity and cell proliferative capacity.
本研究的目的是评估一种针对驴卵巢组织的玻璃化方案，使用由不同浓度（3M或6M）的二甲基亚砜或乙二醇单独组成，或作为组合（3M二甲基亚砜 + 3M乙二醇）的不同冷冻保护剂溶液来保存腔前卵泡。通过视频腹腔镜从巴西东北部品种的母驴获取了十对卵巢，并使用每种冷冻保护剂溶液对皮质碎片进行固体表面玻璃化（SSV）。对卵巢组织进行卵泡形态和活力、DNA完整性（TUNEL技术）以及颗粒细胞中核仁组织区的存在情况（AgNOR技术）的评估。解冻后，与新鲜对照组相比，玻璃化卵巢组织碎片中正常腔前卵泡的百分比显著降低（p < 0.05）。在比较不同处理时，与6M二甲基亚砜（69.8 ± 16.5%）和6M乙二醇（72.3 ± 18.0%；p < 0.05）相比，使用3M二甲基亚砜（81.7 ± 37.5%）、3M乙二醇（83.7 ± 27.4%）以及二者的组合3M二甲基亚砜 + 3M乙二醇（81.8 ± 46.8%）可使更高百分比的卵泡存活。当使用二甲基亚砜 + 乙二醇组合进行玻璃化时，与其他玻璃化处理相比，观察到更高百分比（62.5 ± 29.1%）的活卵泡（台盼蓝）（p < 0.05）。TUNEL技术表明，除了3M二甲基亚砜 + 3M乙二醇处理外，所有测试处理均显示卵泡细胞中有DNA片段化。在评估核仁组织区的存在情况时，使用3M二甲基亚砜 + 3M乙二醇的新鲜组和玻璃化组之间核仁组织区的数量未观察到显著差异（p > 0.05）。我们得出结论，3M二甲基亚砜 + 乙二醇组合对取自马属驴的卵巢组织进行玻璃化更有效，能够充分保存腔前卵泡的形态、活力、DNA完整性和细胞增殖能力。
