Eficacia de tres protocolos de criopreservación de espermatozoides equinos con diferentes velocidades de enfriamiento sobre la calidad espermática postdescongelación

Abstract

The cooling rate determines intracellular and extracellular ice formation and, consequently, sperm cryosurvival. This work evaluated the efficacy of a cryopreservation protocol based on two freezing ramps with accelerating cooling rates compared with two commercial freezing protocols: one conventional with decelerating cooling rates, and another with programmed cooling rates by the biological freezer. For this, 24 ejaculates from 4 sexually adult Arabian stallions were collected with an artificial vagina, diluted with Botusemen Gold® + 5% dimethylformamide, and frozen by exposing the straws (0.5 mL) to liquid nitrogen (LN2) in three different protocols: [1] PCC-1: Conventional (Minitube®) with 1 freezing ramp at 5 cm from the LN2 level for 10 min; [2] PC2R-2: two ramps at 17 cm (for 4 min) and 2 cm (for 2 min) from the level of LN2 content inside a cryobox of 31 x 31 x 30 cm (length, width, and height, respectively); and [3] PCBP-3: programmable biological freezer TK-4000 (Tecnologia®). Ice nucleation and initial cooling rate occurred at -13.8ºC to 63.5ºC/min, at -8.5ºC to 19ºC/min, and at -5.9ºC to -11.4ºC/min for PCC-1, PC2R-2, and PCBP-3, respectively. The cryopreservation process reduced (P<0.01) the kinematic parameters and integrity of sperm membranes, regardless of the protocol used. There were no significant differences (P>0.05) neither kinematics nor plasma membrane integrity between freezing protocols. However, the PC2R-2 and PCBP-3 protocols produced higher post-thaw acrosomal integrity than the PCC-1 protocol. In conclusion, the PC2R-2 protocol produced accelerating cooling rates and cryosurvival of equine spermatozoa similar to commercial protocols of conventional freezing or programmed cooling rates, even improving acrosomal integrity. The use of the two-ramp freezing protocol is recommended as a successful alternative to cryopreserve stallion sperm due to its easy application, efficacy, and less consumption of LN2.