Effect of glycerol concentrations on a new extender for freezing dog semen

Glycerol is the most commonly used cryoprotectant for frozen semen in several species, because it alters colligative properties of water, decreasing freezing point and increasing sperm survival. Many studies used various concentrations of glycerol for freezing dog semen, with no consensus regarding best glycerol concentration. Aim was to evaluate glycerol concentrations (6, 7, and 8%) in dog freezing extender. Two ejaculates from 12 adult dogs (2 - 5 years) of various breeds were used. Ejaculates were collected by digital manipulation and divided into 3 aliquots, centrifuged at 600 x g for 7 minutes and pellet was resuspended (100 x 10 6 sperm per ml) in an egg yolk-based extender BotuDog ® (Botupharma® ) with 3 glycerol concentrations: 6, 7, and 8% (G6, G7, and G8, respectively). Samples were packaged in 0.5 ml straws (IMV, France) and kept in a controlled temperature refrigerator (Minitub® ) for 60 minutes at 5o C. Straws were placed in a Styrofoam box and kept 3.5 cm above nitrogen level for 20 minutes, and finally were immersed. Straws were thawed at 76°C for 8 seconds. Sperm kinetics were evaluated using the CASA system (Hamilton Thorne Research - IVOS 12, Beverly, MA). Integrity of plasma and acrosomal membranes, and stability of plasma membrane were determined by flow cytometry (BD LSR Fortessa - Becton Dickinson, Mountain View, CA), at 10 and 30 minutes (T10 and T30), after thawing with incubation at 37o C. Data were analyzed using statistical program Graph Pad 6.0 and KolmogorvSminov test was used for normality. ANOVA followed by Tukey test were used and Friedman test followed by Dunns were used for nonparametric data. Total motility was not different (p > 0.05) among groups in T10: G6 (52.5 ± 5.3), G7 (58.7 ± 3.7), and G8 (57.3 ± 5.3) and in T30: G6 (35.1 ± 3.9), G7 (41.4 ± 4.1), and G8 (38.2 ± 3.8), neither progressive motility in T10: G6 (39.5 ± 4.9), G7 (45.4 ± 3.8), and G8 (44.1 ± 4.8) and in T30: G6 (26.3 ± 3.7), G7 (32.0 ± 3.8), and G8 (29.7 ± 3.6). Integrity of plasma and acrosomal membrane was higher in G7 (52.6 ± 3.4) compared to G6 (42.4 ± 3.7) at 10 minutes after thawing and after 30 minutes incubation in G7 (32.3 ± 2.8) compared to G6 (26.4 ± 2.7). We concluded that 7% glycerol in the Botudog ® freezing extender yielded better results for plasma and acrosomal membrane integrity than 6%, whereas 8% had intermediate results.

Keywords: Cryopreservation, canine, CASA, plasma and acrosomal membrane integrity

This manuscript was originally published in the journal Clinical Theriogenology Vol 12(3) Sept 2020.  Clinical Theriogenology is the official journal of the Society for Theriogenology (SFT) and the American College of Theriogenologists (ACT).  This content has been reproduced on the IVIS website with the explicit permission of the SFT/ACT.