Evaluation of Fertilizing Potential of Frozen‐thawed dog Spermatozoa Diluted in ACP‐106^®using anIn VitroSperm–Oocyte Interaction Assay

Abstract

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.

Intravaginal Artificial Insemination in Bitches Using Frozen/Thawed Semen after Dilution in Powdered Coconut Water (ACP‐106c)

“…The better results obtained with the current study compared with a previous study using the same extender (Cardoso et al. ) are perhaps because of the reduction in egg yolk concentration from 20% to 10%, thus favouring smaller amounts of lumps and better dissolution, with a consequently improved visualization of sperm cells. The choice to use 10% egg yolk in the present study was based on additional results obtained by our research team (Barbosa et al.…”

“…The better results obtained with the current study compared with a previous study using the same extender (Cardoso et al 2007) are perhaps because of the reduction in egg yolk concentration from 20% to 10%, thus favouring smaller amounts of lumps and better dissolution, with a consequently improved visualization of sperm cells. The choice to use 10% egg yolk in the present study was based on additional results obtained by our research team (Barbosa et al 2009) using ACP-106c, which showed that this lower concentration is more practical for the analysis of sperm motility (objective or computerized) because of the lower viscosity of the medium, which facilitates sperm cell displacement.…”

“…This medium has been used for semen freezing (Cardoso et al. ) and for artificial insemination (AI) with cooled semen in dogs (Uchoa et al. ); however, there are no reports of normal pregnancy when using ACP‐106c with frozen/thawed canine semen.…”

“…A powdered coconut water-based extender ACP-106c (ACP ® ; ACP Servic¸os Tecnolo´gicos Ltda, ACP Biotecnologia, Fortaleza, Ceara´, Brazil) has been developed in recent years. This medium has been used for semen freezing (Cardoso et al 2007) and for artificial insemination (AI) with cooled semen in dogs (Uchoa et al 2012); however, there are no reports of normal pregnancy when using ACP-106c with frozen/thawed canine semen.…”

The effect of swim-up purification and incubation of cells on sperm viability in dogs of different ages

“…Freezing techniques and the evaluation of fertilizing potential of frozen-thawed dog spermatozoa have been intensively investigated in several studies (Zavos et al, 1992;Martins-Bessa et al, 2006;Schafer-Somi et al, 2006Cardoso et al, 2007). However, determining the viability of sperm of dogs of different ages has not been the subject of focus.…”