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

La formación de hielo intracelular y extracelular va a depender de la velocidad de enfriamiento y, en consecuencia, la criosupervivencia espermática. Esta investigación evaluó la eficacia de un protocolo de criopreservación de dos rampas de congelación con velocidades de enfriamiento aceleradas frente a dos protocolos de congelación comercial: uno convencional con velocidades de enfriamiento desaceleradas, y otro con velocidades programadas con congelador biológico. Para esto, 24 eyaculados de 4 caballos Árabes sexualmente maduros fueron recolectados con una vagina artificial, diluidos con Botusemen Gold® + 5% dimetilformamida, y congelados exponiendo las pajuelas (0,5 mL) a nitrógeno líquido (NL2) en tres protocolos distintos: [1] PCC-1: Convencional (Minitube) con 1 rampa de congelación a 5 cm del nivel de NL2 durante 10 minutos; [2] PC2R-2: dos rampas a 17 cm (durante 4 minutos) y 2 cm (durante 2 minutos) del nivel de NL2 mantenidas dentro de un cryo-box de 31 x 31 x 30 cm (largo, ancho y alto, respectivamente); y [3] PCBP-3: congelador biológico programable TK-4000 (Tecnología). La nucleación de hielo y la velocidad de enfriamiento inicial se produjeron a los -13,8ºC a 63,5ºC/min, a - 8,5ºC a 19ºC/min, y a -5,9ºC a -11,4ºC/min, respectivamente en el PCC-1, PC2R2 y PCBP-3. El proceso de criopreservación redujo (P<0,01) los parámetros cinemáticos e integridad de las membranas espermáticas, independientemente del protocolo usado. No se registró diferencias significativas (P>0,05) ni en la cinemática ni en integridad de la membrana plasmática entre protocolos de congelación. Sin embargo, los protocolos PC2R-2 y PCBP-3 produjeron una mayor integridad acrosomal post-descongelación comparado con el protocolo PCC-1. En conclusión, el protocolo PC2R-2 produjo velocidades de enfriamiento aceleradas y una criosupervivencia de espermatozoides equinos similar a los protocolos comerciales de congelación convencional o de velocidades programadas, incluso mejorando la integridad acrosomal. Se recomienda el uso del protocolo de congelación de dos rampas como alternativa exitosa para criopreservar espermatozoides equinos debido a su fácil aplicación, eficacia y menos consumo de NL2.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.Médico Veterinario ZootecnistaCuenc

Optimization of cryopreservation of arabian stallion sperm using dimethylformamide, glycerol, and different freezing protocols

This study evaluated the effect of penetrating cryoprotectant agents (CPA) and the cryosurvival of three freezing protocols on the kinematics and integrity of membranes of frozen-thawed stallion sperm. Twenty-four ejaculates of four adult Arabian horses were collected in six weekly sessions (six ejaculates/horse). Each ejaculate was divided into two aliquots. With the first aliquot, three CPA treatments were conformed: 5% glycerol (GLY), 5% dimethylformamide (DMF), and 3%–3% DMF–GLY combination, and the sperm samples were frozen exposing them to liquid nitrogen (LN2) vapors. The second aliquot was diluted with freezing medium plus 5% DMF and the sperm samples were frozen in three freezing protocols: (P1) Styrofoam cryo-box (30 × 29 × 31 cm of length, width, and height, respectively) with two ramps (at 17 and 7 cm above LN2); (P2) freezing unit® (Minitüb, Germany); and (P3) programmable TK 4000-freezer® (Compacta, Brazil). The DMF-GLY combination and DMF yielded higher (p.05) post-thaw SM, VCL, and IPIA than the other protocols. Indeed, the P1 and P3 protocols yielded lower proportion (p<.05) of sperm with damaged plasma and damaged acrosome than the P2 protocol after thawing (3.7±0.18 and 3.1±0.18 vs. 6.1±0.44%, respectively). In conclusion, the addition of DMF or combined with GLY to freezing medium, and the freezing with Styrofoam cryo-box with two ramps increase the cryosurvival of Arabian stallion spermatozoa.Leó