Master of Science (MS)
School of Animal Science
In this study, I evaluated the cryopreservation by pellet freezing of spermatozoa from individual dogs. In Experiment I, spermatozoa from 15 pairs of epididymides were suspended in glycerol, frozen as pellets of 10, 50, 100, or 200 μl volumes, and thawed by dilution with TALP (Tyrode's solution plus albumin, lactate, pyruvate). In Experiment II, spermatozoa from 16 pairs of epididymides were suspended in glycerol, dimethyl sulfoxide, or ethylene glycol, frozen as 100 μl pellets, and thawed by dilution with TALP, canine capacitation medium (CCM), or 3% sodium citrate solution. In Experiment III, spermatozoa from 15 pairs of epididymides were suspended in glycerol, frozen as 100 μl pellets, and thawed by dilution with CCM. In Experiment IV, ejaculated spermatozoa from each of three dogs and epididymal spermatozoa from each of four other dogs were suspended in glycerol and were frozen and thawed as in Experiment III. Survival was determined by microscopic evaluation of motility and of membrane integrity. In Experiments III and IV, survival was also assayed by measuring the zona-binding capacity of the spermatozoa. Survival of frozen-thawed samples was significantly lower than unfrozen samples. Sperm survival after freezing depended significantly on the pellet volume, reflecting the effect of cooling rate as a function of pellet volume. Thawing solutions and CPAs also significantly affected post-thaw sperm survival. The highest post-thaw survival was obtained with a pellet volume of 100 μl and with the CPA-thawing solution combinations of glycerol-CCM and glycerol-TALP. The number of membrane-intact spermatozoa bound to each oocyte was significantly higher for unfrozen samples than for frozen-thawed samples. Ejaculated spermatozoa exhibited survival and zona-binding capacity similar to that of epididymal spermatozoa, and there was little variation in the survival of ejaculated spermatozoa from an individual dog. There were significant differences in post-thaw sperm survival and zona-binding capacity among individual dogs. These results complement previous studies showing male-to-male differences in freezing susceptibility of canine spermatozoa.
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Fahrig, Brooke, "Cryopreservation by pellet freezing of epididymal and ejaculated spermatozoa from male dogs" (2003). LSU Master's Theses. 1859.