CRYOPRESERVATION OF MOUSE SPERMATOZOA .1. EFFECT OF SEEDING ON FERTILIZING ABILITY OF CRYOPRESERVED SPERMATOZOA

To examine the effect of seeding to induce ice formation during cryopr eservation 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 + g lycerol as cryoprotective additives (CPAs). Seeding and holding sperma tozoa at -4 degrees C did not affect their viability as judged by vita l staining. Egg yolk protected spermatozoa against chilling injury, as cooling them to -4 degrees C in the presence of egg yolk yielded high er survivals than those cooled without egg yolk (34.4 +/- 3.4 v9.0 +/- 1.3% in three replicates of >400 spermatozoa/replicate). To study eff ects of seeding on their fertilizing ability, spermatozoa in the raffi nose-glycerol-egg yolk solution were frozen to -196 degrees C either w ithout seeding or after seeding at -4 degrees C. Development of 222 oo cytes into two-cell embryos after in vitro fertilization (IVF) with sp ermatozoa frozen without seeding was 43%; development rates of 186, 18 6. 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 unseede d spermatozoa the respective cleavage rates into two-cell embryos were 83% of 275 oocytes and 69% of 304 oocytes, a difference that was smal l but significant by X-2 analysis. An additional 925 oocytes were fert ilized with spermatozoa after being seeded and frozen to -196 degrees C in four separate batches of CPA solutions. Overall, after IVF with f rozen 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 met hod described here are capable of fertilizing oocytes at a rather high rate. (C) 1997 Academic Press.