Choi Y H, Love C C, Varner D D, Hinrichs K
Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, TX 77843-4466, USA.
Theriogenology. 2006 Mar 1;65(4):808-19. doi: 10.1016/j.theriogenology.2005.04.035. Epub 2005 Aug 10.
This study was conducted to evaluate the effects of thawing, division into aliquots and refreezing on fertilizing capacity (ability to support embryo development after intracytoplasmic sperm injection; ICSI) of frozen stallion semen. Frozen semen from a fertile stallion was thawed, diluted 1:100 with freezing extender, and refrozen (2F treatment). Control semen was frozen only once. In vitro matured equine oocytes were injected with: (1) motile control spermatozoa; (2) motile 2F spermatozoa; (3) non-motile 2F spermatozoa; or (4) non-motile 2F spermatozoa, followed by injection of sperm extract. Blastocyst development after ICSI was equivalent between control spermatozoa and motile 2F spermatozoa (27 and 23%, respectively). Blastocyst development after injection of non-motile 2F spermatozoa (13%) tended (P=0.07) to be lower than that for control spermatozoa. Injection of sperm extract into oocytes that received non-motile 2F spermatozoa resulted in a significant decrease in blastocyst development (to 2%) compared with injection of non-motile 2F spermatozoa alone. Spermatozoa from a subfertile stallion was similarly processed and used for ICSI; blastocyst development for both motile control (once frozen) spermatozoa and motile 2F spermatozoa was 9%. In conclusion, frozen stallion semen may be thawed, diluted, and refrozen without effect on the ability of motile spermatozoa to initiate embryo development after ICSI. Non-motile spermatozoa from reprocessed semen may also achieve embryo development after ICSI. To our knowledge, this is the first report evaluating the ability of refrozen spermatozoa to produce embryos by ICSI in any species.
本研究旨在评估解冻、分装和再冷冻对冷冻种马精液受精能力（胞浆内单精子注射后支持胚胎发育的能力；ICSI）的影响。将来自一匹可育种马的冷冻精液解冻，用冷冻稀释液按1:100稀释后再冷冻（2F处理）。对照精液仅冷冻一次。将体外成熟的马卵母细胞注射以下物质：(1) 活动的对照精子；(2) 活动的2F精子；(3) 不活动的2F精子；或(4) 不活动的2F精子，随后注射精子提取物。ICSI后的囊胚发育在对照精子和活动的2F精子之间相当（分别为27%和23%）。注射不活动的2F精子后的囊胚发育率（13%）略低于对照精子（P=0.07）。与仅注射不活动的2F精子相比，向接受不活动2F精子的卵母细胞中注射精子提取物导致囊胚发育显著下降（降至2%）。对一匹亚育种种马的精子进行类似处理并用于ICSI；活动的对照（冷冻一次）精子和活动的2F精子的囊胚发育率均为9%。总之，冷冻种马精液可以解冻、稀释和再冷冻，而不会影响活动精子在ICSI后启动胚胎发育的能力。再处理精液中的不活动精子在ICSI后也可能实现胚胎发育。据我们所知，这是第一份评估再冷冻精子通过ICSI在任何物种中产生胚胎能力的报告。
