PARENT SESSION
Epididymis and Sperm Maturation
295
EFFECT OF EXTRACTION METHOD ON CONCENTRATION AND CRYOSURVIVAL OF DOMESTIC DOG EPIDIDYMAL SPERM.
Amodeo, Dianne1, Anderson, Autumn1, Wetsman, Irene1, Olson, Mary 1, Durrant, Barbara1, 1
ABSTRACT- The domestic dog is a convenient model for the development of extraction and cryopreservation techniques for epididymal sperm of endangered carnivores post-mortem. In Experiment 1, 101 epididymides were obtained from 52 dogs following routine castration and assigned to one of four sperm extraction methods: 1) FL, 5 ml of TEST-yolk buffer (T-Y) was flushed from the proximal vas deferens through a series of 5 incisions in the cauda epididymis; 2) MC, the cauda epididymis was minced into T-Y and filtered to separate sperm from tissue; 3) EX, epididymides were manually compressed to extrude sperm through caudal incisions into T-Y; 4) SW, sperm was allowed to swim out for 10 minutes through epididymal incisions into T-Y. Sperm concentrations were determined by hemocytometer count. Each of the 25 epididymides in Experiment 2 was assigned to one of the four extraction methods, avoiding duplication of individual dogs within a treatment. Initial motility score (IMS, calculated as motility x speed of progression2 ) and initial viability (IL, plasma membrane integrity determined by eosin-nigrosin staining) were recorded prior to cooling for 30 min to 4°C. Glycerolated T-Y was then added (for a final concentration of 4% glycerol) and vials of extended sperm were frozen in LN2 vapor. After a minimum of 10 days storage in LN2 , sperm was thawed for 1 min in a 37°C water bath, and %IMS and %IL were evaluated immediately (T0). Sperm was washed by centrifugation, resuspended in M199 medium and assessed for %IMS and %IL after 30 min (T30) and 60 min (T60) at 37°C. The effect of extraction method on sperm concentration, %IMS and %IL was analyzed by ANOVA. Although not statistically significant, EX and MC tended to yield higher concentrations of sperm than SW and FL (p=0.344; EX 128.7 ± 16, MC 125.1 ± 25, SW 96.9 ± 22, FL 77.3 ± 15 x 106/ml). In addition, EX tended to maintain the greatest %IMS at T60 (p=0.069) and resulted in the highest %IL at T30 (p=0.005) and T60 (p=0.006). These data show that manual extrusion provided a high concentration of sperm with the least compromise in post-thaw motility or plasma membrane integrity.
KEY WORDS: epididymis, dog, cryopreservation, sperm