The morphological, physical and fertilization parameters of in vitro matured, preovulatory and ovulated oocytes are similar. However, in vitro matured mammalian oocytes are prone to polyspermic fertilization in vitro. Polynuclear zygotes can cleave and form blastocysts at the same rate as in vivo derived two-pronuclear zygotes. Furthermore, blastocysts derived from polynuclear zygotes can implant/attach at the same rate as their two-pronuclear-derived counterparts. Even though polyspermy does not perturb the development of early stage embryos up to attachment/implantation, the phenomenon contributes to reduced pregnancy rates even when blastocysts are transferred synchronously into the uterus. Under in vivo conditions, fertilization occurs in the estrogen-dominated ampulla of the oviduct. Therefore, estrogen regulated fluid from the ampulla (AOVF) should permit mono-spermic fertilization. Also, the presence of adenosine in IVF reaction mixture has been shown to reduce polyspermic fertilization. Cryopreservation of sperm provides a reliable source for sperm dudng unusual times when non-frozen sperm is not available. The former sperm require activation in most instances and our preliminary data have shown that 2-chloroadenosine (2-CA) is a potent motility activator of frozen-thawed sperm. It is therefore, conceivable that fertilization of mature oocytes with 2-CA-activated sperm in the presence of AOVF, collected at the time of anticipated ovulation, can limit or prevent polyspermic fertilization in vitro. Our long-range goal is to improve IVF conditions, to permit mono-spermic fertilization in vitro. The objective of this project is to study the influence of harvested porcine AOVF at the time of anticipated ovulation and 2-CA, on polyspermic fertilization in vitro. The centr'al hypothesis is that specific factors secreted by the oviduct at the period of estrogen dominance limit or prevent polyspermic fertilization, while the reverse hypothesis is that polyspermic fertilization increases in vitro when IVF is conducted in the absence of oviductal factors. Our hypothesis has been formulated on the basis of strong preliminary data produced in our laboratory. The rationale for the proposed study is that once polyspermic fertilization is reduced during IVF, term pregnancy rates will increase post synchronous embryo transfers (ETs). We plan to test our central hypothesis and accomplish the overall objective of this study with the following specific aims: 1. Determine the effect of porcine AOVF on motion characteristics and pre- fertilization function of 2-CA- activated frozen-thawed porcine and human sperm; 2. Determine the influence of AOVF and 2-CA on zona pellucida sperm binding, pig IVF outcome, and in utero survival of transferred derived pig blastocysts; 3. Determine the generational effects of 2-CA on development.