Poster Presentation IXth International Conference on Boar Semen Preservation 2019

Effect of extender, cryoprotectants and GameteGuard® in post-thaw motility in boar sperm (#1.9)

Raul A. Gonzalez 1 , Amy M. Sharf 1 , Amanda Minton 2 , Lisa A. Herinkhoff 1
  1. Membrane Protect Technologies Incorporated, Fort Collins, CO, USA
  2. The Maschhoffs, St. Louis, MO, USA

The ability of sperm to withstand freeze-thawing is highly variable among boars. Therefore, cryopreservation techniques have to be optimized to reduce cryodamage and increase reproductive performance. We aimed to evaluate the effect of extenders, cryoprotectants (CPA) and GameteGuard® (MPTI, Fort Collins, CO, USA) on post-thaw motility. Two ejaculates from each of six Duroc boars were extended 1:1 (Androstar® Plus, Minitube, Verona, WI, USA) and held at 17 °C for 24 h. Semen was centrifuged at 600 x g for 15 min. The pellet was divided, suspended in a cooling extender [lactose, trehalose, lactose-trehalose (1:1, v/v) or TRIS extender plus 20% egg yolk] with the presence or absence of GameteGuard® (4%, v/v). After cooling (4 °C for 90 min), freezing extender containing glycerol alone (3%) or combined with methylformamide (GMF; 1% glycerol 3% methylformamide) plus 0.5% Equex STM (Minitube) was added, resulting in a final concentration of 600x106 sperm/mL. Cooled semen was packed in 0.5-mL straws, placed 4 cm above liquid nitrogen for 15 min and then plunged into liquid for storage. Three straws per treatment were thawed at 37 °C for 30 s, pooled, diluted (TRIS, 1:20) and evaluated for percentage of total and progressive motility by CASA (IVOS IITM, Hamilton-Thorne, Beverly, MA, USA). Data were examined by mixed model, using a three-factor design with boar as the random effect for repeated measures, and results shown as means±sem. As main effects, percentages of total and progressive motility were not affected by base-extender (P>0.09). However, different cryoprotectants and the use of GameteGuard® influenced (P<0.0001) both motility parameters, with important interactions between base-extender and GameteGuard® for total (P<0.001) and progressive motility (P<0.001), and base-extender with cryoprotectants for total (P<0.001) and progressive (P<0.07) motility. Total motility of sperm was higher when frozen with glycerol (lactose: 38.9±3.5: TRIS: 34.3±3.5), than with GMF (lactose: 26.3±3.5; TRIS:16.7±3.5; P<0.009), or those frozen in trehalose (24.3±3.5; P<0.006) or lactose-trehalose (24.3±3.5; P<0.006). Total motility in extender containing trehalose was similar for glycerol and GMF (P>0.8). When sperm were frozen with GameteGuard® in different extenders, total motility was higher (P<0.02) compared to controls (lactose: 34.4±3.5 vs 26.2±3.5; trehalose: 29.1±3.5 vs 22.1±3.5; trehalose-lactose: 32.7±3.5 vs 18.8±3.5). Sperm frozen with GameteGuard® and either glycerol (31.3±3.2) or GMF (29.1±3.2) displayed similar total motility (P>0.3), but both higher than those frozen with GMF without GameteGuard® (19.5±3.2; P<0.0001). After analyzing the main effects and interactions among the main components of the freezing extenders for boar sperm, we conclude that lactose-based extender with glycerol resulted in higher post-thaw motility compared to TRIS or trehalose-based extenders or the use of methylformamide as the cryoprotectant. The addition of GameteGuard® improved post-thaw motility in the lactose-based extender with glycerol, and may provide a method for commercialization of the frozen extender.