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Submitted to: Journal of Animal Science
Publication Type: Abstract Only Publication Acceptance Date: 5/14/2004 Publication Date: 7/25/2004 Citation: Purdy, P.H. 2004. Effects of holding time prior to freezing on the motility, viability and membrane binding ability of ram sperm. 2004 Joint Annual Meeting of the American Dairy Science Association, American Society of Animal Science and Poultry Science Association, July 26-28, 2004, St. Louis, Missouri. Journal of Animal Science. 82(Suppl 1):367. Interpretive Summary: The United States sheep industry lacks infrastructure to effectively collect and store genetic resources in the national repository. Therefore, we investigated a methodology that could be used to ship diluted ram semen samples that were cooled and held at 5ºC for up to 48 hours prior to cryopreservation. Semen samples from 6 rams were collected and the concentration and motility were determined. Samples were diluted with a one-step Tris-egg yolk-glycerol media and cooled to 5°C over 2 hours using a styrofoam shipping box and commercial cold packs. The samples were maintained at 5°C in the shipping box, and aliquots were loaded into 0.5 mL French straws at 0, 24 or 48 hours after cooling, frozen in liquid nitrogen and plunged for storage. No differences between freeze times (0, 24, 48 h) were detected using ANOVA in post thaw motility, plasma membrane integrity, or live acrosomal. No differences were observed in the mean number of cells binding to chicken oocyte perivitelline membranes at time 0, 24 and 48 h. These results indicate that ram sperm may be held at 5ºC for up to 48 hours prior to freezing with no deleterious effects on post thaw motility, plasma membrane integrity and acrosomal integrity. In addition, the chicken oocyte membrane binding assay demonstrates a simple in vitro method to assess post-thaw ram sperm capacitation, acrosome reaction and binding ability. The combination of the shipping protocol and viability testing has the potential to ease the constraint on collecting and freezing ram semen. Technical Abstract: The United States sheep industry lacks infrastructure to effectively collect and store genetic resources in the national repository. Therefore, we investigated a methodology that could be used to ship diluted ram semen samples that were cooled and held at 5ºC for up to 48 hours prior to cryopreservation. Semen samples from 6 rams were collected and the concentration and motility were determined using spectrophotometry and computerized automated semen analysis (CASA), respectively. Samples were diluted to 400 x 10**6 cells per mL with a one-step Tris-egg yolk-glycerol media and cooled to 5°C over 2 hours using a styrofoam shipping box and commercial cold packs. The samples were maintained at 5°C in the shipping box, and aliquots were loaded into 0.5 mL French straws at 0, 24 or 48 hours after cooling, frozen in vapor 4.5 cm above liquid nitrogen for 12 to 13 minutes and plunged for storage. No differences between freeze times (0, 24, 48 h) were detected using ANOVA in post thaw motility (29, 31, 36%; P > 0.05), plasma membrane integrity (28, 35, 29%; P > 0.05) or live acrosomal integrity (99, 99, 99%; P > 0.05). Motility was assessed using CASA, and plasma membrane integrity and acrosomal integrity were simultaneously determined using the fluorescent stains propidium iodide and FITC-PNA, respectively, with flow cytometry. In addition, no differences were observed in the mean number of cells binding to a chicken oocyte membrane (461, 532, 319; P > 0.05) at time 0, 24 and 48 h, respectively. These results indicate that ram sperm may be held at 5ºC for up to 48 hours prior to freezing with no deleterious effects on post thaw motility, plasma membrane integrity and acrosomal integrity. In addition, the chicken oocyte membrane binding assay demonstrates a simple in vitro method to assess post-thaw ram sperm capacitation, acrosome reaction and binding ability. The combination of the shipping protocol and viability testing has the potential to ease the constraint on collecting and freezing ram semen. |
