Thesis Title

The Effects Of Equine Spermatozoa/Oocyte Co-Culture On Spermatozoal Acrosome Reaction And Viability

Date of Graduation

Summer 2001


Master of Science in Biology



Committee Chair

Jane Pruitt

Subject Categories



The present study was conducted to determine the influence of oocyte and spermatozoal interactions on acrosome reaction and viability of equine spermatozoa during in vitro fertilization. Three ejaculates were collected from three stallions on an every other day basis. Semen was processed and allowed to incubate in 37⁰ C with 5% CO₂ incubator for 15 minutes. At the completion of the incubation period, the sample was divided. One portion was returned to incubate for 4.5 hours. A viability stain, Hoechst 33258, was added to the second portion. Three groups of six salt-stored oocytes were inseminated with stained spermatozoa. Spermatozoa and oocytes were allowed to co-culture for one minute. At one minute, three oocytes from each group were removed and placed in new media to allow for continued co-culture with closely associated spermatozoa. Paraformaldehyde was added to the one-minute co-culture to halt all reactions and fix spermatozoal membranes. Spermatozoa were removed from oocytes and stained for acrosome reaction. Spermatozoa were evaluated by fluorescent microscopy for percent acrosome reaction and percent viability. Sixty-minute co-cultured spermatozoa were processed and evaluated as previously stated for the one-minute co-culture and the 4.5-hour incubated spermatozoa were processed and evaluated as previously stated for the 15-minute incubation. Spermatozoa co-cultured for prolonged periods (1 min. vs. 60 min.) had increased percent acrosome reaction (P<0.0001) and also demonstrated an increase in percent acrosome reaction by lengthened incubation time (P<0.0449). Percent viability of spermatozoa for all groups decreased over time (P<0.0001) of incubation, however, percent viability was not different among treatments (P=0.8461). Therefore, this study indicates that incubation time and co-culture significantly increased percent acrosome reaction. However, co-culture had no effect on increase of viability and viability was significantly reduced over incubation time.


© Gavin O'Connor