Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4 degrees C until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 microM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.

Purpose Cryopreservation of blastocysts, especially those subjected to the trauma due to blastomere biopsy for the purposes of pre-implantation genetic screening (PGS), requires significant optimization. Laboratory and clinical outcomes were compared to determine the effect of two different cryopreservation techniques on the development of human pre-implantation embryos that underwent blastomere biopsy and blastocoel drainage prior to cryopreservation. Design Retrospective clinical study. Patient(s) Women who requested cryotransfer of supernumerary blastocysts were analyzed by FISH. Results The main outcome measures were post-thaw survival (SR), pregnancy (PR), and implantation (IR). The SR of slowly frozen blastocysts was 83% compared to 97% for vitrified blastocysts. In 160 cases where biopsied embryos were cryotransferred, the results for slowly frozen versus vitrified blastocysts were: SR (71% vs. 95%), PR (23% vs. 37%), and IR (26% vs. 36%, P<0.05), respectively. Conclusion The results revealed that vitrified blastocysts provided higher SR, PR and IR as compared to slowly frozen counterparts.

A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

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