Evaluation of Fertilizing Potential of Frozen-thawed dog Spermatozoa Diluted in ACP-106^® using an In Vitro Sperm–Oocyte Interaction Assay

Contents

The aim of present study was to evaluate frozen canine semen with ACP-106^® (Powder Coconut Water) using an in vitro sperm–oocyte interaction assay (SOIA). Ten ejaculates from five stud dogs were diluted in ACP-106^® containing 20% egg yolk, submitted to cooling in a thermal box for 40 min and in a refrigerator for 30 min. After this period, a second dilution was performed using ACP-106^® containing 20% egg yolk and 12% glycerol. Samples were thawed at 38°C for 1 min. Post-thaw motility was evaluated by light microscopy and by using a computer aided semen analysis (CASA). Plasma membrane integrity and sperm morphology/acrosomal status were evaluated by fluorescent probes (C-FDA/PI) and Bengal Rose respectively. Moreover, frozen-thawed semen was analysed by a SOIA. Subjective post-thaw motility was 52.0 ± 14.8% and it was significant higher than the total motility estimated by CASA (23.0 ± 14.8%) because this system considered the egg yolk debris as immotile spermatozoa. Although normal sperm rate and acrosomal integrity evaluated by Bengal Rose stain was 89.6 ± 3.1% and 94.3 ± 3.1%, respectively, post-thaw percentage of intact plasma membrane was only 35.1 ± 14.3%. Regarding SOIA, the percentage of interacted oocytes (bound, penetrated and bound and/or penetrated) was 75.3%. Using regression analysis, it was found significant relations between some CASA patterns and data for SOIA. In conclusion, the freezing-thawing procedure using ACP-106^® was efficient for maintain the in vitro fertility potential of dog spermatozoa.