Use of polyvinyl alcohol as a chemically defined compound in egg yolk‐free extender for dog sperm cryopreservation

Abstract

The objectives of this study were to investigate the effects of polyvinyl alcohol (PVA) as a chemically defined compound in egg yolk (EY)‐free extender by determining the appropriate concentration of PVA and the effect of pH adjustment in EY‐free PVA extenders on dog spermatozoa. Spermatozoa (1 × 108 cells/ml) were frozen with EY‐free extenders supplemented with 0 (control), 0.025, 0.05, 0.1, 0.2 or 0.3 g/100 ml PVA. Sperm progressive motility (PM) was assessed immediately after thawing (IAT) and post‐thaw incubation (PTI), while viability, acrosome integrity and reactive oxygen species (ROS) levels were evaluated after PTI. Additionally, spermatozoa were frozen using EY‐free PVA extenders before pH adjustment (6.45) and after adjustment of pH (6.85). Viability, PM, ROS and gene expression (BCL2 and SMCP) were assessed. Supplementation with 0.05 g/100 ml or more PVA significantly increased PM compared to the control group in the IAT and PTI. Post‐thaw incubation significantly increased sperm motility in all groups. The acrosome integrity in all PVA groups was higher (p < .05) than the control without an effect on ROS and viability. Adjustment of the pH to 6.85 improved (p < .05) sperm PM compared to the non‐adjusted groups without affecting viability, ROS or expression of BCL2 and SMCP. We suggest that PVA supplementation in EY‐free Tris extenders can effectively protect dog spermatozoa during freezing and can maintain higher motility and acrosome integrity. Adjustment of pH in EY‐free PVA extenders can improve post‐thaw sperm motility. Therefore, PVA can be used as a compound in EY‐free extender for the cryopreservation of dog spermatozoa.

The Use of κ-Carrageenan in Egg Yolk Free Extender Improves the Efficiency of Canine Semen Cryopreservation

“…Unless otherwise noted, all chemicals used in the present study were purchased from Sigma-Aldrich (St. Louis, MO, USA).The extenders used in the present study were extender 1 which consists of 2.42% [ v/v ] Tris, 1.34% [ v/v ] citric acid, 0.8% [ v/v ] glucose, 0.06% [ v/v ] penicillin, and 0.1% [ v/v ] streptomycin in distilled water (Invitrogen, Carlsbad, CA, USA) and various κ-carrageenan concentrations (0.0%, 0.1%, 0.2%, 0.3%, and 0.5%). Extender 2 was composed of extender 1 plus 9.2% [ v/v ] glycerol [ 8 ].…”

“…Numerous approaches have been attempted to improve the quality of frozen-thawed (FT) sperm. The addition of cryoprotectant agents such as glycerol, egg yolk, milk, polyvinyl alcohol, and cholesterol to extenders have been reported to reduce the detrimental effects of the freezing and thawing process [ 6 , 7 , 8 , 9 , 10 , 11 ]. Since its discovery as a component of cooling extender, egg yolk has been widely used in mammalian sperm cryopreservation to protect sperm from initial cold shock [ 12 , 13 ].…”

Assisted reproductive techniques for canines: preservation of genetic material in domestic dogs

“…Polyvinyl alcohol (PVA) is an attractive alternative to egg yolk. Although no comparative experiment has been conducted with the extender containing egg yolk, approximately 35% of progressive motility has been reported in cryopreserved spermatozoa that used an extender with PVA [ 55 ]. Further studies are needed to confirm the fertility of these spermatozoa upon artificial insemination.…”

Cryopreservation of Siberian tiger (Panthera tigris altaica) epididymal spermatozoa: pilot study of post-thaw sperm characteristics

“…Sperm freezing was performed following (Nabeel et al, 2019). Briefly, spermatozoa (10 × 10 6 sperm/mL) were first suspended in extender 1 (20 mM trehalose supplemented Norwegian extender: 3.025 g Tris, 1.7 g citric acid, 1.25 g fructose, 0.06 g penicillin, 0.1 g streptomycin, 20% egg yolk (EY) in 100 mL distilled water) and incubated in 4℃ for 1 h. After that, extender 2 (extender 1 with 10% glycerol) was added to the extended sperm (final concentration: 5 × 10 6 sperm/mL).…”