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Equine Sperm Membrane Phase Behavior: The Effects of Lipid-Based Cryoprotectants¹

Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine;³ Electron Microscopy Laboratory, Department of Pathology, School of Medicine;⁴ Veterinary Medical Teaching Hospital;⁵ Center for Biostabilization;⁶ Section of Molecular and Cellular Biology;⁷ Department of Population Health and Reproduction, School of Veterinary Medicine,⁸ University of California, Davis, California 95616

ABSTRACT

The plasma membrane of sperm can undergo lipid phase separation during freezing, resulting in irreversible damage to the cell. The objective of our study was to examine the membrane phase behavior of equine spermatozoa in the absence and presence of lipid-based cryoprotectants. Biophysical properties of sperm membranes were investigated with Fourier-transform infrared spectroscopy. Compared to fresh untreated sperm, postthaw untreated sperm showed extensive lipid phase separation and rearrangement. In contrast, postthaw sperm that were cryopreserved in egg phosphatidylcholine (egg PC)- or soy phosphatidylcholine (soy PC)-based diluents showed similar lipid phase behavior to that of fresh, untreated sperm. Studies with a deuterium-labeled PC lipid (POPCd-31) suggest that exogenous lipid from the diluents are strongly associated with the sperm membrane, and scanning electron microscopy images of treated sperm show the presence of lipid aggregates on the membrane surface. Thus, the exogenous lipid does not appear to be integrated into the sperm membrane after cryopreservation. When compared to a standard egg-yolk-based diluent (INRA 82), the soy and egg PC media preserved viability and motility equally well in postthaw sperm. A preliminary fertility study determined that sperm cryopreserved in the soy PC-based medium were capable of fertilization at the same rate as sperm frozen in the conventional INRA 82 medium. Our results show that pure lipid-based diluents can prevent membrane damage during cryopreservation and perform as well as a standard egg-yolk-based diluent in preserving sperm viability, motility, and fertility.

gamete biology, sperm, sperm motility and transport

¹ Supported through the UC Davis Center for Equine Health, with funds provided by the Oak Tree Racing Association, the State of California Pari-Mutuel Fund, and contributions by private donors, as well as USDA Cooperative State Research, Education and Extension Service NRI grant 2002–35203–12260.

² Correspondence: S.A. Meyers, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, UC Davis, One Shields Ave., Davis, CA 95616. FAX: 530 752 7690; smeyers{at}ucdavis.edu