374 THE EFFECTS OF POLYVINYLPYRROLIDONE ON BOVINE SPERM FUNCTION AND EMBRYO DEVELOPMENT

Abstract

In assisted reproduction by ICSI, PVP has been successfully used to replicate the viscosity of sperm solution, thus facilitating the handling and immobilization of spermatozoa. Sperm is suspended in medium containing polyvinylpyrrolidone (PVP), then injected into the oocytes together with a small amount of the medium in ICSI. However the effects of PVP on sperm function and embryo development have not been investigated in detail. In the present study, we investigated the effects of PVP solution on sperm function and embryonic development. Frozen–thawed spermatozoa from a Japanese Black bull and immature COCs from slaughterhouse bovine ovaries were used for all experiments. In experiment 1, bovine sperm was cultured in SOF or SOF containing 10% PVP. For detection of sperm acrosomal and chromatin integrity, sperm cultured in each medium were stained by the triple staining method and acridine orange after 0, 15, 30, and 60 min of culture. In experiment 2, zygotes were injected with PVP solution and cultured in vitro; subsequent cleavage and development to blastocysts were examined. In experiment 3, zygote injected with PVP solution was fixed by 4% paraformaldehyde after 1–3 h of PVP injection. The location of PVP solution in zygote was observed. In experiment 4, two-cell embryos were microinjected with a solution of dextran conjugated with fluorescein (FITC-dextran) and cultured in vitro. The location of FITC-dextran in the embryo was examined. In experiment 1, acrosome reactions of the sperm were enhanced after 15 min of incubation in PVP solution (P < 0.05), but chromatin integrity of the sperm was not influenced (P > 0.05). In experiment 2, PVP suppressed the development of the zygote to 2-cell, morula and blastocyst (75.0%, 35.1%, and 26.3% vs. 61.3%, 20.2%, and 12.9% for control and PVP group, respectively, P < 0.05). In experiment 3, the locations of PVP solution in the zygote were observed 1–3 h after injection. In experiment 4, FITC-dextran was observed in ICM at the blastocyst stage. These findings suggest that PVP affects the acrosome but not the chromatin of sperm in ICSI. PVP solution exists locally in embryos injected and affects the developmental capacity of the embryos.