ABSTRACTS, 24th ANNUAL This rate was highly dependent on freezing technique and cryoprotective diluent but not dilution ratio. Using a standardized equilibration environment, optimal results were achieved using either straw approach compared to the pelleting method. The manual freezing of straws over liquid nitrogen vapor was comparable to the use of an automated, programmable liquid nitrogen unit. Although the BFSF and TEST diluents resulted in similar motility ratings immediately post-thaw, BFSF, an extender containing a surfactant component, reduced cellular damage and sustained sperm motility longer than TEST. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. In this study, this damage was illustrated by an increased loosening of the acrosomal cap, a factor which did not appear sensitive to any of the freezing methods tested. (Funded, in part, by Friends of the National Zoo). 101. The Effect of One Step and Gradual Reduction in Glycerol Concentration after Thawing on the Survival of Ram Spermatozoa. P. S. FISERAND
R. W. FAIRFULL (Animal Research Centre, Agriculture Canada, Ottawa, Ontario, Canada KlA OC6). Ram semen, collected by artificial vagina, was diluted and processed for long term storage as described by Fiser (Cryobiology 23, 518-524). Concentration of the cryoprotectant, glycerol, was adjusted to 4% in the diluted semen prior to freezing by one step addition at 30°C (Method I), by cooling the semen to 5°C and addition of the glycerol gradually over 30 min (Method 20, or in a one step (Method 3) prior equilibration for 2 hr, or by cooling to SC, followed by a holding period of 2 h at 5°C and the one step addition of glycerol just prior to freezing (Method 4). After thawing, the semen was subjected to removal of glycerol by either a gradual decrease in concentration and osmolality (stepwise dilution from 4% to 0.4% over 15 or 30 mitt), or by a one-step tenfold dilution. The overall post-thaw percentage of motile spermatozoa (range 43.7-44.2%, means pooled over method of glycerination) and the percentage of intact acrosomes (range 56.8-59.5%) did not differ significantly in semen subjected to gradual decrease in glycerol concentration and diluent osmolality (over 15 and 30 min) or by a one step tenfold dilution. However the postthaw percentage of motile spermatozoa was significantly lower after addition of glycerol by Method 1 (39.8%) than when the glycerol was added by the other three methods (range 44.0-46.4%). The postthaw percentage of intact acrosomes was highest (61.2%) in semen in which the glycerol was added by Method 2. This percentage was not significantly different from those in which glycerol was added to
semen by Methods 3 and 4, but it was significantly higher than that found in semen in which the glycerol was added by Method 1 (54.4%). These data indicate that post-thaw spermatozoa integrity can be maintained even after a rapid decrease in glycerol concentration such as accompanies insemination or dilution of semen for assessment of motility. 108. Cryopreservation of Human Semen. ER-TONG CHEN,’ TSE-CHAO HuA,~ YA-WEN XIN,’ YING DA-MING,~ AND YANG RONG~(‘Cryobiological Engineering Laboratory, Shanghai Institute of Mechanical Engineering, Shanghai, China; and 2Shanghai Research Institute of Pediatrics, Shanghai, China). Although cryopreservation of human semen has been reported, we studied the comparison of different methods. The actual temperatures in the semen samples were measured during freezing and thawing using a new cryomicroscope system that had a computer to control temperature and a video tape to record the semen’s behavior during freezing and thawing. The effects of cryopreservation, not only survival but also grade, were examined when adding different concentrations to KS or glycerol as cryoprotective agents. We found that KS was a little better than glycerol, both KS and glycerol would injure human sperm at unfrozen states. The suitable cooling rates are 0.6”Cimin to 7”C/min in the region of 0°C to -40°C; the suitable warming rates are 5O”Clmin or more. In order to get these cooling and warming rates, we introduced three methods: freezing by the programmable freezer; a two-step cooling method using a refrigerator; and a two-step cooling method using a LN, Dewar. The two-step cooling method using a refrigerator was preferable, especially because of its simplicity. 109. Cryopreservation
and Cryobiological Study of the Spermatozoa of the Giant Panda (Alluroposa Melanoseusa). JINSHAN YANG et al.
(Beijing Medical University and Beijing Zoo, Beijing, China). The semen specimens from three giant pandas were cryopreserved successfully and reversibly at - 196°C for breeding purposes and for study of cryobiology, cytochemistry, and ultrastructure. Glycerol, 4.7- 15%, 5- 15% DMSO, and yolk were used as the cryoprotectant. The spermatozoa were cooled and frozen by means of the program Cell Freezer (Planer R 204, England), freezing plates of polyfluortetraethylene (PFE) freezing vials by the vapor of liquid nitrogen. The recovery, mobility, DNA, acrosome, PAS positive substance, and some enzymes and ultrastructure of the cryopreserved spermatozoa were tested and studied at 1 day, 1 week, and every month after the cryopreser-