Adjusting cryodiluent composition for improved post-thaw quality of rabbit spermatozoa

Improved fertility following artificial insemination with frozen-thawed spermatozoa would offer rabbit producers faster genetic improvement. Previous work investigating cryoprotectants for rabbit spermatozoa have reported inconsistent results. Semen was collected from three rabbit bucks by artificial vagina and frozen using a standard procedure with varied cryodiluent components. Post-thaw analysis encompassed motility, sperm kinematic parameters and acrosome and membrane integrity. Spermatozoa were evaluated at 0, 2 and 4 h after thawing. Experiment 1 compared diluents with 3.5% dimethyl sulfoxide (DMSO), 1.5% acetamide, 1.75% DMSO + 0.75% acetamide or 3.5% DMSO + 1.5% acetamide. The treatment that resulted in the highest post-thaw motility (P<0.001) and acrosome integrity (P<0.001) was DMSO alone. Experiment 2 compared 3.5, 7 and 10% DMSO in the cryodiluent. The best post-thaw sperm motility (P<0.001) and linearity (P=.002) was in 3.5% DMSO, while 10% DMSO afforded higher acrosome/membrane integrity at this last time point (P<0.05). Experiment 3 varied the cryodiluent to contain either 9 or 17% egg yolk or 9 or 17% low density lipoproteins extracted from whole egg yolk. The treatment with the best post-thaw result was 17% egg yolk (motility, P=0.01; acrosome/membrane integrity, P<0.001). Experiment 4 compared different carbohydrates in the cryodiluent; 50 mM glucose (TCG), 25 mM glucose with 25 mM sucrose (TCGS low), or 50 mM glucose with 50 mM sucrose (TCGS high). When data were pooled across time points, TCG had significantly higher motility than TCGS high (P=0.021), but was not different from TCGS low. However, TCG had fewer spermatozoa with intact acrosomes and membranes than both TCGS low and TCGS high (P=.002). Put together, these results indicate that the best cryodiluent for rabbit spermatozoa frozen under the conditions used in this paper is with 7% DMSO and 17% egg yolk in a base medium containing 25 mM glucose and 25 mM sucrose.