PARENT SESSION
PLATFORM SESSION 13: ART 2002: AN EMBRYONIC SPACE ODYSSEY
Chair: Pukazhenthi, Budhan¹, ¹
Co-chair: Navara, Christopher¹, ¹
Harborside D
2:00 PM-4:00 PM

(244) INTRACYTOPLASMIC SPERM INJECTION IS MORE EFFICIENT THAN IN VITRO FERTILIZATION FOR CRYOPRESERVED SPERMATOZOA.

Szczygiel, Monika¹, Kusakabe, Hirokazu¹, Whittingham, David¹, Yanagimachi, Ryuzo¹, ¹ Institute for Biogenesis Research, Honolulu, HI

ABSTRACT- Efficient and dependable mouse cryopreservation methods are urgently needed because the production of mice with transgenes, disrupted genes, and mutant genes is now commonplace. Preservation of these unique genomes provides an essential safeguard for future research. Unfortunately mouse spermatozoa appear more vulnerable to freezing than other species e.g. bovine and human. In this study we examined the efficiency of ICSI and IVF in generating embryos from mouse spermatozoa frozen with 18% raffinose and 3% skim milk for cryoprotection. A comparison was made between the inbred strain C57BL/6J, commonly used in mutagenic and transgenic studies, and a hybrid strain B6D2F1 (C57BL/6J x DBA/2J). C57BL/6J spermatozoa are known to be more sensitive to freezing than B6D2F1. Fertilization of oocytes after IVF was significantly lower with C57BL/6J spermatozoa when compared with B6D2F1 spermatozoa for both fresh and frozen spermatozoa (fresh - 89% vs 55%: frozen - 56% vs 9%). Freezing also reduced the fertility of B6D2F1 spermatozoa (89% vs 56%). Fertilization improved dramatically after ICSI with fresh and frozen C57BL/6J spermatozoa (90% and 85%) and also with frozen B6D2F1 spermatozoa (87%). The development of 2-cell embryos to the blastocyst stage was lower for C57BL/6J than for B6D2F1 (42-61% vs. 84-98%) in all treatments, but similar for embryos within each strain. The normality of chromosomes from fresh and frozen spermatozoa was assessed in oocytes prior to first cleavage in both strains. The majority of oocytes had normal chromosomes after IVF (98-100%) and ICSI (87-95%) indicating that chromosomal abnormalities were not responsible for the poorer development in vitro of C57BL/6J embryos. In conclusion, our data show that ICSI is a more efficient and effective technique than IVF for generating embryos from frozen spermatozoa. More importantly, ICSI is especially valuable for strains where IVF with fresh spermatozoa produces few or no embryos. (This work is funded by the National Institute of Child Health and Human Development [NICHD] and NCRR, NIH Grant No.U0 1HD38205 and is part of the National Program on Mouse Sperm Cryopreservation)

KEY WORDS: in vitro fertilization, sperm, embryo, gamete biology