Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic 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 withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was 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 distilled water (297 to 19 mosm/kg H₂O) and stained to assess membrane integrity with Calcein-AM (CAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI stained dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined 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, were detected in 0% PBS (61.1 ± 4.8, P < 0.05). The percentages of swollen 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, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 °C in PBS or H₂O (Trial II). The percentage of viable spermatozoa 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). Subjecting in vitro-stored sperm cells to hypo-osmotic stress before fluorescent staining resulted in detection of labile spermatozoa not accounted for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.