Evaluation of Fertilizing Potential of Frozen-Thawed Dog Spermatozoa Diluted in ACP-106 Using an in Vitro Sperm--Oocyte Interaction Assay

Reprod Domest Anim. 2007 Feb;42(1):11-6. doi: 10.1111/j.1439-0531.2006.00703.x.


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 degrees 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acrosome / physiology
  • Animals
  • Cryopreservation / veterinary*
  • Cryoprotective Agents / pharmacology*
  • Culture Media / chemistry
  • Dogs* / physiology
  • Flow Cytometry
  • Male
  • Semen Preservation / methods
  • Semen Preservation / veterinary*
  • Sperm Motility
  • Sperm-Ovum Interactions / physiology*
  • Spermatozoa / physiology*


  • Cryoprotective Agents
  • Culture Media