Songsasen N, Leibo S P
Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1, Canada.
Cryobiology. 1997 Nov;35(3):240-54. doi: 10.1006/cryo.1997.2048.
To examine the effect of seeding to induce ice formation during cryopreservation on their survival, spermatozoa from B6D2F1 mice were cooled to and held at -4 degrees C for 30 min in phosphate-buffered saline (PBS) alone, in egg yolk-supplemented PBS, or in PBS with raffinose + glycerol as cryoprotective additives (CPAs). Seeding and holding spermatozoa at -4 degrees C did not affect their viability as judged by vital staining. Egg yolk protected spermatozoa against chilling injury, as cooling them to -4 degrees C in the presence of egg yolk yielded higher survivals than those cooled without egg yolk (34.4 +/- 3.4 v 9.0 +/- 1.3% in three replicates of >400 spermatozoa/replicate). To study effects of seeding on their fertilizing ability, spermatozoa in the raffinose-glycerol-egg yolk solution were frozen to -196 degrees C either without seeding or after seeding at -4 degrees C. Development of 222 oocytes into two-cell embryos after in vitro fertilization (IVF) with spermatozoa frozen without seeding was 43%; development rates of 186, 186, and 207 oocytes after IVF with spermatozoa frozen after seeding and being held at -4 degrees C for 5, 10, or 30 min were 46, 44, and 9%, respectively. In a direct comparison, after IVF with seeded or unseeded spermatozoa the respective cleavage rates into two-cell embryos were 83% of 275 oocytes and 69% of 304 oocytes, a difference that was small but significant by chi2 analysis. An additional 925 oocytes were fertilized with spermatozoa after being seeded and frozen to -196 degrees C in four separate batches of CPA solutions. Overall, after IVF with frozen sperm, 68% of those oocytes cleaved into two-cell embryos and 59% developed into 544 blastocysts. Based on these results, we concluded that embryo production by IVF seemed to be improved using spermatozoa frozen after being seeded. Mouse spermatozoa cryopreserved by the method described here are capable of fertilizing oocytes at a rather high rate.
为了研究冷冻保存过程中接种诱导冰晶形成对精子存活的影响，将B6D2F1小鼠的精子在单独的磷酸盐缓冲盐水（PBS）、添加蛋黄的PBS或含有棉子糖+甘油作为冷冻保护剂（CPA）的PBS中冷却至-4℃并保持30分钟。通过活体染色判断，在-4℃接种并保存精子不会影响其活力。蛋黄可保护精子免受冷损伤，因为在有蛋黄存在的情况下将精子冷却至-4℃，其存活率高于无蛋黄冷却的情况（在三个重复实验中，每个重复>400个精子，分别为34.4±3.4%和9.0±1.3%）。为了研究接种对精子受精能力的影响，将棉子糖-甘油-蛋黄溶液中的精子在不接种或在-4℃接种后冷冻至-196℃。用未接种冷冻的精子进行体外受精（IVF）后，222个卵母细胞发育为二细胞胚胎的比例为43%；接种后在-4℃保存5、10或30分钟再冷冻的精子进行IVF后，186、186和207个卵母细胞的发育率分别为46%、44%和9%。在直接比较中，用接种或未接种的精子进行IVF后，二细胞胚胎的各自分裂率分别为275个卵母细胞中的83%和304个卵母细胞中的69%，通过卡方分析，这一差异虽小但具有显著性。另外925个卵母细胞在四个单独批次的CPA溶液中接种并冷冻至-196℃后用精子受精。总体而言，用冷冻精子进行IVF后，68%的卵母细胞分裂为二细胞胚胎，59%发育为544个囊胚。基于这些结果，我们得出结论，使用接种后冷冻的精子进行IVF似乎可提高胚胎产量。用本文所述方法冷冻保存的小鼠精子能够以相当高的比率使卵母细胞受精。
