Pregnancy rates following artificial insemination with frozen-thawed jack spermatozoa have been relatively low compared to those attained in other species. Cholesterol is known to interfere with post-thaw fertility of jack and stallion semen. Altering the amount of cholesterol in the freezing extender may improve the fertility of frozen-thawed jack semen. Here we report clinical work completed with semen collected from a single jack; extended in EZ Mixin ^® OF and slowly cooled to 5°C. Extended semen samples were then centrifuged at 400G for 10 minutes and the supernatant removed. The spermatozoa were frozen in liquid nitrogen vapor after resuspension in the appropriate freezing medium to a final concentration of 400 x 10⁶ cells/mL. Freezing extender treatments were: (1) 20% Egg yolk (EY); (2) 5% EY; and (3) 20% EY + 60mM hydroxypropyl-β-cyclodextrin (β-CD). A total of 28 mares 2 to 18 years in age were utilized over 5 breeding seasons (82 total cycles). Mares were administered human chorionic gonadotropin (hCG) to induce ovulation when the dominant follicle was ≥35 mm as assessed by ultrasonography and were inseminated within 6 hours pre-ovulation and again within 6 hours post-ovulation. Pregnancy rates for each treatment were as follows: (1) 6.25% (1 pregnancy, 15 matings), (2) 46.5% (20 pregnancies, 43 matings), (3) 58.5% (14 pregnancies, 24 matings). These data support the theory of cholesterol interfering with post-thaw fertility of jack semen. We have established that mule pregnancies can be achieved at acceptable rates with frozen-thawed jack semen cryopreserved in 5%EY and 20% EY + 60 mM β-cyclodextrin transferred directly post-thaw.

Cyclodextrins are reported to improve post-thaw viability and motility in boar semen. Cyclodextrin mediates cholesterol efflux and subsequent acrosome reaction and capacitation in the sperm cells of several species in vitro. The second objective of this study was to evaluate whether or not addition of hydroxypropyl-β-cyclodextrin to freezing extenders containing low or high concentrations of egg yolk (cholesterol source) would improve laboratory indicators of fertility for jack and stallion semen and to evaluate post-thaw effects of cyclodextrin on acrosome reaction. Post-thaw motility was improved (p<.05) for jack semen cryopreserved in freezing extender containing 20% egg yolk and 60 mM β-cyclodextrin compared to 5% egg yolk freezing extender, with all other treatments for jack and stallions being similar. Post-thaw viability was not different for species or freeze treatments. Post-thaw addition of cyclodextrin to jack and stallion sperm for 90 minutes induced the acrosome reaction 93.5±5.94% and 22.5 ± 4.66% of viable cells, respectively, as measured by a triple stain procedure and subsequent analysis via flow cytometry. Here we have demonstrated that hydroxy-propyl-β-cylcodextrin can be utilized as a powerful agent of induction of acrosome reaction for the jack and stallion semen.