Yong Hwan Yul, Hong Ji Young, Kang Sung Keun, Lee Byeong Chun, Lee Eun Song, Hwang Woo Suk
Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul 151-742, South Korea.
Theriogenology. 2005 Feb;63(3):783-94. doi: 10.1016/j.theriogenology.2004.05.004.
The present study investigated the correlation of sperm movement in the ooplasm, pretreatment of sperm with dithiothreitol (DTT) and sperm freezing with the development of porcine embryos derived from modified intracytoplasmic sperm injection (ICSI). In vitro, matured gilt oocytes without centrifugation were injected with head membrane-damaged spermatozoa aspirated tail-first. In Exp. 1, frozen-thawed sperm were categorized into three groups: impaired, immotile or motile. Oocytes injected with motile sperm (43.6%) showed a higher (P < 0.05) fertilization rate compared to oocytes injected with impaired or immotile sperm (34.5 or 37.2%). The survival rate was significantly higher (P < 0.05) in oocytes injected with impaired sperm (92.9%) than in oocytes injected with immotile or motile sperm (84.8 or 86.7%). No differences were observed in the rates of cleavage or blastocyst formation, and in total cell number of blastocysts among three groups of oocytes. In Exp. 2, motile frozen-thawed sperm were pretreated with DTT before injection and non-treated sperm served as controls. Higher rates (P < 0.05) of fertilization, male pronucleus (MPN) and decondensed sperm head (DSH) formation were observed in oocytes injected with control sperm (41.1, 50.0 and 91.1%, respectively) than in oocytes injected with DTT-treated sperm (22.1, 30.2 and 72.1%, respectively). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In Exp. 3, motile frozen-thawed or fresh sperm without DTT pretreatment were injected into oocytes. The rates of fertilization and MPN formation were significantly higher (P < 0.05) in oocytes injected with fresh sperm (59.8 and 73.5%) than in oocytes injected with frozen-thawed sperm (36.7 and 59.2%). No differences in embryo development and total cell number of blastocysts were observed between two groups of oocytes. In conclusion, the present study clearly demonstrated that sperm movement in the ooplasm, use of DTT and fresh spermatozoa did not significantly affect on embryo development in porcine modified ICSI.
本研究调查了卵质内精子运动、用二硫苏糖醇（DTT）对精子进行预处理以及精子冷冻与经改良胞浆内单精子注射（ICSI）获得的猪胚胎发育之间的相关性。在体外，将未离心的成熟后备母猪卵母细胞注射尾先吸入的头部膜受损精子。在实验1中，冻融精子被分为三组：受损、不动或有活力。与注射受损或不动精子的卵母细胞（分别为34.5%或37.2%）相比，注射有活力精子的卵母细胞（43.6%）显示出更高（P<0.05）的受精率。注射受损精子的卵母细胞（92.9%）的存活率显著高于注射不动或有活力精子的卵母细胞（分别为84.8%或86.7%）（P<0.05）。三组卵母细胞在卵裂率、囊胚形成率以及囊胚总细胞数方面未观察到差异。在实验2中，有活力的冻融精子在注射前用DTT进行预处理，未处理的精子作为对照。与注射经DTT处理精子的卵母细胞（分别为22.1%、30.2%和72.1%）相比，注射对照精子的卵母细胞（分别为41.1%、50.0%和91.1%）观察到更高（P<0.05）的受精率、雄原核（MPN）形成率和去浓缩精子头部（DSH）形成率。两组卵母细胞在胚胎发育和囊胚总细胞数方面未观察到差异。在实验3中，将未用DTT预处理的有活力的冻融或新鲜精子注射到卵母细胞中。与注射冻融精子的卵母细胞（分别为36.7%和59.2%）相比，注射新鲜精子的卵母细胞（59.8%和73.5%）的受精率和MPN形成率显著更高（P<0.05）。两组卵母细胞在胚胎发育和囊胚总细胞数方面未观察到差异。总之，本研究清楚地表明，卵质内精子运动、DTT的使用和新鲜精子对猪改良ICSI中的胚胎发育没有显著影响。
