Abstract
Gamete preservation of wild species is a priority in conservation programs. However, field techniques to recover sperm from deceased animals may impair its final quality.The cryopreservation process also has its challenges, and the minimal required quality parameters may differ depending on the intended use of the semen.Often, postmortem harvested and cryopreserved sperm may need to undergo a selection process to improve the final quality of the sample. Certain sperm selection techniques that employ centrifugal forces can increase the risk of damaging the cells. In contrast, microfluidics uses hydrostatic pressure and capillary forces, avoiding the need for costly equipment and minimizing cell damage.This study aims to compare sperm motion parameters, morphology, and acrosome integrity of cryopreserved-thawed epididymal bighorn ram sperm using two commercial selection techniques, VetCount Harvester® device (MFD) and BovipureTM (NidaCon International AB, Möndal, Sweden) density gradient centrifugation (DGC).Sperm parameters associated with quality were, progressive motile sperms (PMS), straight velocity (VSL), average path velocity (VAP), curvilinear velocity (VCL), linearity (LIN), straightness (STR), beat cross frequency (BCF), amplitude of lateral head deviation (ALH) and plasma membrane integrity (PMI). Computer Assisted Sperm Analyzer was used for the semen analysis. Eosin/nigrosine and Spermac stains were used to assess morphology and acrosome integrity, respectively. Two veterinarians counted the total number of morphologically normal sperm with complete acrosome.Mean sperm concentration was higher (P < 0.01) before filtration (129.56 ±53.9 ×106/ml) than DCG (68.42 ±29.18 ×106/ml) and MFD (52.94 ±28.76 ×106/ml). PMS was higher (P <0.01) after selection using the DGC (79.70 ± 11.11) and MFD (81.23 ±5.64) compared to the no sperm-selected controls (45.36 ±14.67). Sperm kinetics parameters (VCL, VSL, VAP, BCF, LIN, and STR) were higher in the DGC (P<0.01). However, ALH (P<0.01) was significantly higher in the MFD and Control. Morphological normal sperm, acrosome integrity, and plasma membrane integrity were higher (P<0.01) in samples processed with DGC and MFD.In conclusion, microfluidics and single-layer centrifugation improved motion parameters, viability, and proportion of morphologically normal sperm with intact acrosomes from frozen-thawed sperm harvested from Bighorn epididymis. The VetCount Harvester® microfluidic device was comparable to DGC and could be an alternative option for sperm selection. Further research is needed to explore the effect of selected sperm using this technique on artificial insemination, re-cryopreservation, and fertility outcomes.
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