ASSESSMENT OF THE MEMBRANE INTEGRITY OF FRESH AND STORED TURKEY SPERMATOZOA USING A COMBINATION OF HYPOOSMOTIC STRESS FLUORESCENT STAINING AND FLOW-CYTOMETRY

A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of w ithstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial wa s repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in d istilled water (297 to 19 mosm/kg H2O) and stained to assess membrane integrity with Calcein-AM (GAL) and propidium iodide (PI). The CAL det ected viable spermatozoa (green fluorescence) while the PI shined dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determ ined by flow cytometry, was not different from that in 100% PBS (76.4/-3.8) to 20% PBS (74.1+/-3.5). Fewer viable spermatozoa, however, wer e detected in 0% PBS (61.1+/-4.8, P < 0.05). The percentages of swolle n tails observed for viable (green stained) spermatozoa were 0, 4.5, 6 .5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respective ly. Semen was also evaluated fresh or after 24 h in vitro storage at 5 degrees C in PBS or H2O (Trial II). The percentage of viable spermato zoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H2O (48.0+/-5.1) than in PBS (66.1+/-5.6, P<0.05). Subjec ting in vitro-stored sperm cells to hypo-osmotic stress before fluores cent staining resulted in detection of labile spermatozoa not accounte d for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.