61 Swim-up and microfluidic techniques improve the kinetic parameters of selected bovine spermatozoa for in vitro fertilization: Preliminary results

For invitro embryo production, spermatozoa with fertilizing capacity must possess optimal kinetic, morphometric, vitality, and DNA integrity characteristics. The objective of this study was to evaluate the effect of 3 sperm selection methods on sperm quality and invitro embryonic development in bovine. Frozen commercial semen (0.5 mL/straws) from one bull with known fertility was thawed at 37°C for 20 s and was divided for 3 sperm selection techniques: density gradient, swim-up, and microfluidic sperm sorting. The sperm kinetic parameters (VCL = curvilinear velocity, VSL = straight line velocity, VAP = average path velocity, ALH = lateral displacement of sperm head, BCF = beat frequency cross, STR = path straightness) were assessed using computer-assisted sperm analysis (CASA). Sperm morphometric parameters were evaluated using Diff-Quick staining followed by automated analysis. To assess vitality, the sperm were stained with propidium iodide and acridine orange, then analysed under a fluorescence microscope. In addition, DNA fragmentation was assessed using sperm chromatin dispersion method. Last, the fertilizing capacity of the selected sperm was tested by fertilizing cumulus–oocyte complexes (2 × 106 sperm mL−1) obtained from slaughterhouse ovaries and matured invitro for 24 h. A standardized invitro embryo production protocol was used with commercial medium from Vitrogen. The cleavage rate and blastocyst yield were measured on Day 2 and 7, respectively (fertilization = Day 0). The results were calculated with analysis of variance and Tukey’s test (P   0.05) between the 3 sperm selection techniques: density gradient (84.0%), swim-up (75.2%), and microfluidic sorting (67.3%). However, blastocyst yield was significantly higher (P < 0.05) using sperm selected by density gradient (28.1%) and swim-up (21.9%) compared with microfluidic sorting (15.3%). In conclusion, sperm selection using microfluidic and swim-up techniques improved kinetic parameters with lower levels of DNA fragmentation, without affecting sperm morphometry. However, both the density gradient and swim-up techniques are efficient systems for producing invitro bovine embryo.