Department of Veterinary Morphophysiology, Center of Agricultural Sciences, Federal University of Piauí, Teresina, PI, Brazil.
Theriogenology. 2011 Sep 15;76(5):933-41. doi: 10.1016/j.theriogenology.2011.04.024. Epub 2011 Jun 30.
The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.
目的是比较不同的玻璃化技术和溶液在保存包埋于卵巢组织中的山羊窦前卵泡的形态和活力方面的效率。通过常规玻璃化（CV）在法国吸管中、在大管（MTV）中或在固体表面玻璃化（SSV）中冷冻保存卵巢皮质碎片。测试了 6 种含有 6 M 乙二醇的溶液，分别含有或不含有蔗糖（SUC；0.25 或 0.50 M）和/或 10%胎牛血清（FCS）（实验 I）。1 周后，样品被加热，窦前卵泡进行组织学检查。为了评估卵泡活力（实验 II），使用上述三种技术对卵巢碎片进行玻璃化处理，溶液中含有 0.25 M SUC 和 10% FCS。加热后，通过台盼蓝排斥试验评估卵泡。在实验 III 中，使用产生最高比例有活力的窦前卵泡的玻璃化方案对包埋于卵巢组织中的窦前卵泡进行玻璃化处理（含有 0.25 M SUC 和 10% FCS 的 SSV）。加热后，将包埋于卵巢组织中的窦前卵泡进行体外培养，然后通过组织学和荧光显微镜（钙黄绿素-AM 和 ethidium homodimer-1）进行分析。与对照组（88.0%）相比，每种玻璃化方案都显著降低了形态正常的卵泡百分比（88.0%）；然而，在玻璃化溶液中添加 0.25 M SUC 和 10% FCS 改善了卵泡形态的保存（分别为 CV、MTV 和 SSV 的 67.4%、67.4%和 72.0%）。尽管 SSV 后卵泡活力（80.7%）与新鲜（未玻璃化）卵巢组织无差异（88.0%），但体外培养后，有活力的卵泡百分比显著降低（70.0%）。玻璃化卵巢组织体外培养后形态正常的卵泡百分比与未经先前玻璃化处理的卵巢皮质片段培养后的百分比相似（76.0%）（83.2%）。总之，使用含有 0.25 M SUC 和 10% FCS 的 SSV 是山羊卵巢组织玻璃化的最有效方法。
