Abstract:
Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following AI, are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPA), and temperatures on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In Experiment 1, electro-ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900, and 1200 mOsm kg-1 sucrose solutions, held for 5 min, and then returned to isosmotic conditions. Motility characteristics of sperm during exposure to each anisosmotic solution and after return to isosmotic conditions were determined. In Experiment 2, electro-ejaculated and epididymal ram sperm were exposed to 1 <span class="sc-ex">m</span> glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG), or propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after return to isosmotic conditions were determined. In Experiment 3, effects of various temperatures on motility characteristics of electro-ejaculated and epididymal ram sperm were determined after exposure to three different sub-physiologic temperatures (4, 10, and 22�C) for 30 min and subsequently return to 37�C. A computer-assisted semen analysis system was used to determine sperm motility characteristics. Epifluorescence microscopy was used to determine sperm acrosomal integrity after fixing and staining with Alexa Fluor-488-PNA (Molecular Probes, Eugene, OR, USA). The data were analyzed by ANOVA by using SAS (SAS Institute, Inc., Cary, NC, USA). The motility of electro-ejaculated ram sperm was significantly more affected by anisosmotic stress than was that of epididymal ram sperm (P < 0.05). While anisosmotic stress had no effect on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for electro-ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1 <span class="sc-ex">m</span> Gly, DMSO, EG, or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P > 0.05). However, there was a slight decrease in acrosomal integrity for electro-ejaculated ram sperm after exposure to 1 <span class="sc-ex">m</span> Gly, DMSO, or EG (P > 0.05). Both epididymal and electro-ejaculated ram sperm exhibited temperature-dependent loss of motility and acrosomal integrity (P < 0.05). However, electro-ejaculated ram sperm were more sensitive to chilling stress than were epididymal sperm (P < 0.05). In conclusion, current data overall suggest that while epididymal ram sperm are extremely resilient to various cryobiologically relevant stress conditions, ejaculated ram sperm demonstrate greater sensitivity to such stressors. These current findings should be taken into account when handling and developing cryopreservation protocols for ejaculated and epididymal ram sperm.