title: Importance of cooling rate and animal variability for boar sperm cryopreservation: insights from the cryomicroscope.
abstract: A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.
authors.affiliation: Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street, London NW1 0TU, UK. amedrano@servidor.unam.mx
authors.forename: A
authors.initials: A
authors.lastname: Medrano
authors.forename: P F
authors.initials: PF
authors.lastname: Watson
authors.forename: W V
authors.initials: WV
authors.lastname: Holt
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002470:Cell Survival; D020285:Cryoelectron Microscopy; D015925:Cryopreservation / Q000379:methods*; D008297:Male; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility; D013094:Spermatozoa / Q000648:ultrastructure*; D018709:Statistics, Nonparametric; D013552:Swine; D013696:Temperature