Effect of post-thaw addition of seminal plasma to cryopreserved epididymal equine spermatozoa

Abstract

Seminal plasma (SP) has been reported to increase the immediate motility of frozen-thawed epididymal sperm and appears to be important for plasma membrane remodeling, capacitation, acrosomal reaction and fertilization. The objective of this study was to evaluate the effect of incubating equine epididymal frozen-thawed sperm with different concentrations of seminal plasma. Sperm samples were obtained from testes after castration of 40 young Silla Argentino stallions (24 to 36 months of age). Testicles were cooled (5°C) and after 24 h, the cauda epididymis were dissected and flushed (retrograde) with Kenney extender. Epididymal sperm were cryopreserved in EDTA-glucose with 5% DMF, 11% lactose, 20% egg-yolk and Equex, using a rapid temperature-descent curve. The samples were thawed at 37°C for 1 min and then incubated 10 minutes with different concentrations of SP (10%, 50%, 100%) vs. a control (0% SP). To create a pool of SP, Silla Argentino stallions of proven fertility were used. Seminal plasma was separated and stored at -18 °C until use. Semen parameters evaluated were sperm motility (Computer Assisted Semen Analysis; ISAS v1, Proiser), morphology (Diff Quick stain), viability and membrane function (combined hypoosmotic test and Coomassie blue stain). Data were analyzed using Kruskal Wallis test with P<0.05considered significant. Post-thaw incubation with increasing concentrations of seminal plasma significantly decreased sperm total and progressive motility (0%: 33.06 ± 2.02; 26.71 ± 1.90; 10%: 24.06 ± 1.74; 18.85 ± 1.59; 50%: 19.44 ± 1.71; 14.92 ± 1.53; 100%: 14.97 ± 1.57; 11.66 ± 1.48 respectively. Values are mean +/- SEM). However, the values of Beat-Cross Frequency, Linearity and Straightness were significantly higher when the spermatozoa were incubated with SP (BCF: 0%: 10.24 ± 1.47a; 10%: 10.67 ± 3.13a,b; 50%: 10.63 ± 2.31b; 100%: 11.45 ± 2.73b; STR: 0%: 0.59 ± 0.07a; 10%: 0.62 ± 0.09a,b; 50%: 0.64 ± 0.09b; 100%: 0.64 ± 0.12b; LIN: 0%: 0.23 ± 0.05a; 10%: 0.25 ± 0.06b; 50%: 0.26 ± 0.07b; 100%: 0.26 ± 0.07b). There was no effect on membrane function, acrosome status and sperm morphology, regardless of the concentration of SP used (P>0.05). The results indicate that there is no evidence of a beneficial effect of post-thaw supplementation with SP at the concentrations used in this study. It has to be considered that the main role of SP is not associated with sperm physiology but as a modulator in the female reproductive tract. Hence we are continuing in vivo studies to assess whether SP addition can improve pregnancy rates after artificial insemination with frozen-thawed epididymal sperm.