The effects of oocyte storage and cumulus cell presence on canine zona penetration by domestic dog spermatozoa

Zona penetration assays (ZPAs) have been developed in numerous species to evaluate sperm fertilizing potential. Preservation methods to stockpile oocytes would be beneficial because of the difficulty in obtaining sufficient numbers of fresh oocytes. Using a canine ZPA, the objectives of this study were to evaluate: (1) two methods of storing canine oocytes (salt storage and intrafollicular cooling) and (2) the effects of cumulus cells on oocyte penetration. In experiment 1, oocytes from fresh ovaries were assigned at random to 3 categories: fresh control (FRE), salt storage in solution 1 (1.5 M MgCl2(.)6H2O; SS1) and salt storage in solution 2 (0.5 M (NH4)2SO4, 0.75 M MgCl2(.)6H2O, 0.2 mM ZnCl2; SS2). Each category was subdivided into two treatments: cumulus cells intact (intact) and cumulus cells removed (denuded), resulting in a total of six treatments with n greater than or equal to 15 oocytes per treatment for each ejaculate. Fresh (FRE) intact oocytes demonstrated greater sperm-oocyte interaction than other treatments, including FRE denuded oocytes (11.7 +/- 0.6 versus less than or equal to 4.1 +/- 0.5 sperm-oocyte and 94.9 versus less than or equal to 55.6% penetration; P < 0.01). Poor sperm-oocyte interaction was demonstrated with all salt-stored oocytes (less than or equal to 1.6 +/- 0.2 sperm-oocyte and <51% penetration), but was further attenuated in the absence of cumulus cells. In experiment 2, oocytes obtained from fresh (FRESH) or cooled (24 h COOL, 48 h COOL) ovaries were used with cumulus cells intact for a total of three treatments with n greater than or equal to 15 oocytes per treatment for each ejaculate. No significant difference was observed in sperm interaction between oocytes from fresh, 24 and 48 h COOL ovaries (12.3 +/- 0.5 to 13.1 +/- 0.4 sperm-oocyte and 92.2-97.7% penetration; P > 0.01). These results indicate that salt storage may cause damage to canine oocytes, subsequently impairing sperm penetration, whereas short-term intrafollicular cooling does not affect the oocyte's penetrability. Furthermore, greater sperm interaction in oocytes with an intact cumulus suggests a possible role for cumulus cells in canine gamete interaction.