Zhang Y Z, Zhang S C, Liu X Z, Xu Y Y, Wang C L, Sawant M S, Li J, Chen S L
Department of Marine Biology, Ocean University of Qingdao, Qingdao 266003, PR China.
Theriogenology. 2003 Sep 15;60(5):989-96. doi: 10.1016/s0093-691x(03)00097-9.
A simple and convenient protocol for the cryopreservation of the flounder (Paralichthys olivaceus) sperm was established for "on the spot" cryopreservation of large quantities of semen. The use of three cryoprotectants, dimethyl sulphoxide (DMSO), glycerol (Gly) and methanol was tested in the method. The percentage of motile sperm present in semen after it had been frozen and thawed in the presence of DMSO, Gly or methanol was 60.5+/-3.6, 79.17+/-4.5 and 13.25+/-4.7%, respectively. The fertilization rates of this sperm were 67.06+/-15.1, 76.20+/-10.0 and 44.93+/-22.6%, while the hatching rates of eggs fertilized with this sperm were 37.40+/-8.3, 48.18+/-25.7 and 23.35+/-10.8%, respectively. It was found that Gly and DMSO were better cryoprotectants than methanol, with Gly giving the best overall results. Under scanning electron microscopy, it could be seen that while the majority of the frozen-thawed sperm remained morphologically normal, some exhibited lost or dilated mitochondria, swollen mid-pieces, broken tails, or damaged cell membrane, which probably caused the decrease in motility and fertility of the frozen-thawed sperm.
建立了一种简单便捷的牙鲆（Paralichthys olivaceus）精子冷冻保存方法，用于大量精液的“现场”冷冻保存。该方法测试了三种冷冻保护剂，即二甲基亚砜（DMSO）、甘油（Gly）和甲醇。在DMSO、Gly或甲醇存在下冷冻和解冻后的精液中，活动精子的百分比分别为60.5±3.6%、79.17±4.5%和13.25±4.7%。该精子的受精率分别为67.06±15.1%、76.20±10.0%和44.93±22.6%，而用该精子受精的卵子的孵化率分别为37.40±8.3%、48.18±25.7%和23.35±10.8%。结果发现，Gly和DMSO作为冷冻保护剂比甲醇更好，其中Gly的总体效果最佳。在扫描电子显微镜下可以看到，虽然大多数冻融后的精子在形态上保持正常，但有些精子出现线粒体丢失或肿胀、中段肿胀、尾部断裂或细胞膜受损，这可能导致了冻融后精子活力和受精能力的下降。
