Evaluación de dos métodos de selección espermática por centrifugación y flotación de esperma canino epididimario fresco y criopreservado

El presente estudio evaluó dos métodos de selección de espermatozoides epididimarios de perros, uno por centrifugación en gradientes de densidad usando BoviPure (CGD-BoviPure), y otro por Swim-up. Se recuperaron muestras espermáticas de sesenta epidídimos mediante flujo retrógrado usando el diluyente TCG-YH (tris, ácido cítrico, glucosa + 20% de yema de huevo) y se conformaron veinte pools (3 muestras/pool). La selección espermática se realizó en muestras frescas individuales (n=60) o muestras agrupadas (n=20), ya sea frescas, refrigeradas (24 h) o congeladas-descongeladas (C/D). En cada muestra, un grupo control (sin seleccionar) fue incluido. Todas las muestras fueron evaluadas su cinética-morfometría y la integridad de las membranas espermáticas mediante el sistema CASA, (SCA) y la prueba de fluorescencia PI/PNA-FITC, respectivamente. Los resultados demostraron que CGD-BoviPure fue más efectivo en seleccionar espermatozoides de perro que el Swim-up y control, basado en incremento de la cinética e integridad de membranas plasmática (IMP) y acrosomal (IMA). En muestras individuales, la selección por CGD-BoviPure incrementó (P0,05) las dimensiones morfométricas de la cabeza comparados con sus valores antes y después de la congelación. En conclusión, la selección mediante CGD-BoviPure fue exitosa debido a que mejoró la calidad de los espermatozoides epididimarios caninos frescos y criopreservados.The present study evaluated two dog epididymal sperm selection methods, one by BoviPure® density-gradients centrifugation, and the other by conventional swim-up. Sperm samples from sixty epididymides were recovered by retrograde flushing using TCG-EY extender (tris, citric acid, glucose + 20% egg yolk), and twenty pools were conformed (3 samples/pool). The sperm selection procedures were performed on individual fresh samples (n=60) or pooled samples (n=20), either fresh, chilled (24 h), or frozen-thawed (F/T). In each sample, a non-selected control group was included. In all samples, the kinetic-morphometry and sperm membranes integrity were evaluated by the CASA system (SCA®) and PI/PNA-FITC fluorescence test, respectively. The results showed that BoviPure-DGC was more effective in selecting canine sperm than Swim-up and control, based on improving kinetics and integrity of plasma (IPM) and acrosomal (IAM) membranes. In individual samples, the selection by BoviPure-DGC increased (P 0.05) the morphometric dimensions of the head compared with its values before and after freezing. In conclusion, the sperm selection by BoviPure-DGC was successful in improving the quality of fresh and cryopreserved dog epididymal sperm.Médico Veterinario ZootecnistaCuenc

BoviPure® density-gradient centrifugation procedure enhances the quality of fresh and cryopreserved dog epididymal spermatozoa

BoviPure® is a salt solution containing colloidal silica particles coated with silane used to select sperm (e.g., ruminants) by density-gradient centrifugation (DGC). This research assessed the suitability of the BoviPure-DGC and swim-up methods for selecting dog epididymal sperm in fresh, chilled and frozen-thawed samples on post-treatment sperm quality. Sperm samples (n = 60 epididymides) were recovered by retrograde flushing from thirty orchiectomized adult dogs. Thereafter, 20 sperm pools, containing sperm aliquots of three randomly selected animals, were used for chilling (at 5 ºC for 24 h) and freezing (in liquid nitrogen vapors). Sperm selection by BoviPure-DCG and swim-up was performed in both individual and pooled samples, including non- selected samples as controls. Overall, after BoviPure-DGC selection a higher sperm retrieval rate was obtained than the swim-up selection in both individual (P < 0.05) and pooled (P < 0.01) samples. BoviPure-DGC improved (P < 0.05) the total (TM) and progressive (PSM) sperm mo- tilities, curvilinear (VCL) and straight-line (VSL) velocities, linearity (LIN), wobble (WOB), beat- cross frequency (BCF), and integrity of plasmatic (IPM) and acrosomal (IAM) membranes of in- dividual samples in comparison with non-selected samples. In pooled samples, however, the BoviPure-DGC improved (P < 0.05) the PSM, VCL, WOB, and IPM of chilled and frozen-thawed samples. The swim-up method improved (P < 0.05) only some kinematic variables of the indi- vidual (VCL, WOB and BCF) and cryopreserved pooled samples (VCL and ALH) in comparison with non-selected samples. In conclusion, BoviPure-DGC was more effective for recovering and selecting both fresh and cryopreserved dog epididymal sperm than the swim-up procedure improving the kinematic variables, and membranes intactnes