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The Effect of Cryopreservation with and without Seminal Plasma on the Motility and Membrane Integrity of Epididymal Sperm from Feral Boars

Amber C. Williams, D Bruce Lawhorn DVM MS, Dickson Varner DVM DACVT, Kevin J Rozeboom PhD, Brian C Day BS


Objective: Using epididymal harvested spermatozoa from feral boars, the cryoprotective capacity of 5 % domestic boar seminal plasma (SP) in a flushing/incubation medium alone, in combination with 5 % SP in a cooling and freezing extender, or with 5 % SP in a cooling and freezer extender only were compared.

Procedures: Within 12 hours of collection from a feral swine slaughter plant, testicle/epididymal pairs were packed atop wet ice in a cooler, transported to a lab, then flushed. Spermatozoa commingled from each epididymis of a pair were considered one sample. Six samples (D-I) had three treatment groups: (1) Control (no SP exposure); (2) Exposure of spermatozoa to 5 % SP initially in flushing/incubating medium only; and (3) Exposure of spermatozoa to 5 % SP initially in flushing/incubating medium and to 5% SP in the cooling and freezing extender. Three additional samples (A-C) had two treatment groups: (1b) No exposure to SP in flushing/incubating medium and exposure to 5% SP in the cooling and freezing extenders and; (2b) Exposure to 5 % SP in flushing/incubating medium and cooling and freezing extenders. All samples were collected into 0.5 ml straws and frozen in liquid nitrogen. All straws were submitted to a commercial laboratory for assessment of post thaw motility and progressive motility by computer-assisted sperm motility analysis (CASA); plasma membrane integrity was assessed using a dual stain of permeate nucleic acid stain SYBR 14 combined with propidium iodide. For samples D - I, two straws per treatment were evaluated (6 straws total per sample). For samples A and B, two straws for treatment 1b and 4 straws per treatment 2b, respectively, were evaluated (6 straws total per sample).

Results: The post-incubation motility was subjectively assessed and ranged from 40 - 90 % motile with a 63 % average. Total spermatozoa in each sample ranged from 0.73 to 16.8 billion with 5.96 billion average. Sample D came from the largest boar, with the largest testicles and the most volume of spermatozoa collected. The post-thaw motility and membrane integrity of D was the best overall of all samples evaluated. Treatment 3 (Table 2) showed the highest motility in 5/6 samples, the highest progressive motility in 3/6 samples and the greatest plasma membrane integrity in 4/6. Treatment 2b for sample A (Table 3) was higher for all three parameters. Treatment 2b for sample B (Table 3) was higher for motility, lower in progressive motility and the same for plasma membrane integrity. Evaluations were not performed on sample C.

Conclusions: The difference in cryopreservation capacity from exposure of epidiymal spermatozoa to 5 % SP in flushing/incubating medium alone (treatment 2), in combination with exposure to 5 % SP in cooling and freezing extender (treatment 3 and 2b), or with exposure to 5% SP in a cooling and freezing extender only (treatment 1b) seemed superior with treatments 3 and 2b. The final interpretation is pending statistical analysis. A larger sample size should be used in further studies to examine possible differences. Also treatment groups 1, 1b, 2 and 3 (3 and 2b are the same) should be included in any future sample comparisons. The post thaw fertility rate of feral swine spermatozoa is still in question since no artificial insemination was performed. However, these data demonstrate that feral swine spermatozoa seem to be fertile post thaw as described in this experiment.