Efecto crioprotector de la dimetilformamida, glicerol y su combinación sobre la criosupervivencia de espermatozoides equinos

La combinación de agentes crioprotectores penetrantes (ACP) como el glicerol (GLY) y la dimetilformamida (DMF), ha demostrado resultados alentadores en la criosupervivencia de espermatozoides equinos. En este sentido, esta investigación evalúo el efecto crioprotector del GLY, la DMF y su combinación, DMF-GLY, sobre la criosupervivencia de espermatozoides equinos. Para este propósito, se recolectó 24 eyaculados de cuatro caballos Árabes, sexualmente adultos y sanos (6 eyaculados / reproductor). Cada eyaculado fue diluido inicialmente con el diluyente BotusemenSpecial y luego congelado con el diluyente Botusemen-Gold suplementado con GLY (5%), DMF (5%) y GLY-DMF (3%-3%) usando vapores de nitrógeno líquido (NL2) estático. Las muestras espermáticas frescas y congeladas-descongeladas fueron evaluadas sus variables cinemáticas y la integridad de membranas mediante el sistema computarizado CASA (SCA-Evolution-2018) y prueba de doble tinción fluorescente (PI / PNA-FITC), respectivamente. Los resultados demostraron que el DMF sólo o combinado con GLY (DMF-GLY) fue más efectivo para proteger a los espermatozoides equinos y producir mayores valores (P < 0,05) de motilidad total (MT,%) y progresiva (MP,%), velocidad curvilínea (VCL), amplitud del desplazamiento lateral de la cabeza (ALH) y frecuencia de batido de flagelo (BCF) en comparación con el GLY. De hecho, la combinación de DMFGLY produjo una mayor (P < 0,05) velocidad rectilínea (VSL) y promedio (VAP), integridad de la membrana plasmática (IMP, %) y acrosomal (IMA,%) en comparación con el DMF o GLY solos. En conclusión, la combinación de DMF-GLY suplementada al diluyente de base no sintética Botusemen-Gold fue más eficaz para crioproteger a los espermatozoides de caballo Árabe basado en una mayor cinemática e integridad de membranas en comparación que el glicerol.The combination of cryoprotective penetrating agents (CPA), such as glycerol (GLY) and dimethylformamide (DMF), has shown encouraging results in equine sperm cryosurvival. In this sense, this research evaluated the cryoprotective effect of GLY, DMF, and its combination: DMF-GLY, on equine sperm quality and cryosurvival. For this purpose, twenty-four semen ejaculates were collected from four healthy, sexually adult Arabian horses (6 ejaculates/stallion). Each ejaculate was initially diluted with BotusemenSpecial extender and then frozen with Botusemen-Gold extender supplemented with GLY (5%), DMF (5%), and DMF-GLY (3%-3%) using static liquid nitrogen vapors. The fresh and frozen-thawing sperm samples were evaluated for their kinematics variables and membranes integrity using the CASA computerized system (SCA-Evolution-2018) and the double fluorescent test (PI / PNA-FITC), respectively. The results showed that DMF alone or combined with GLY (DMF-GLY) was more effective in protecting equine spermatozoa and producing higher values (P< 0,05) of total (MT, %) and progressive (MP, %) motilities, curvilinear velocity (VCL), the amplitude of lateral head displacement (ALH) and beat-cross frequency (BCF) compared to GLY. The combination of DMF-GLY yielded higher (P < 0.05) strain-line (VSL) and average path velocities (VAP), plasma membrane integrity (IMP, %), and acrosomal membrane integrity (IMA, %) compared to DMF alone or GLY alone. In conclusion, the combination of DMF-GLY supplemented to the 'Botusemen-Gold' non-synthetic-based extender, was more suitable for cryoprotecting Arabian horse sperm based on higher kinematics and membrane integrity compared to glycerol cryoprotectant agent.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