Comparison of Fertility Between Intracytoplasmic Sperm Injection and In Vitro Fertilization with a Partial Zona Pellucida Incision by Using a Piezo-Micromanipulator in Cryopreserved Inbred Mouse Spermatozoa

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.