207 Investigating the Cryotolerance of Boar Spermatozoa Subjected to Prior Selection

Abstract

The proportion of non-viable spermatozoa in insemination semen doses poses limitations to breeding. Recent studies have shown the potential of removing moribund spermatozoa from semen doses through their interactions with specifically designed magnetic nanoparticles (MNP). This specific retrieval leads to substantial enrichment of insemination doses with robust spermatozoa (nanoselected), which would have advantageous effects on the animal husbandry industry. Here, we explored the potential of these MNP for semen dose enrichment and freezability improvement. The MNP were synthesised to target acrosome reacted (lectin) and apoptotic (annexin-V) spermatozoa. Freshly harvested semen of at least 2 boars were pooled and extended in a commercial diluent at a local stud. Extended semen (40 mL) were mixed with varying amounts of MNP (0, 87.5, and 175 μg) and incubated at 37°C for 60 min. Thereafter, the tube mixtures were placed under a magnetic field to trap moribund spermatozoa bound to MNP. The intact or MNP-free (nanoselected) spermatozoa were eluted by pouring the untrapped spermatozoa into new tubes. Both fresh non-selected (control) and nanoselected spermatozoa were mixed with the cryoguard-cooling extender and equilibrated for 120 min at 4°C, followed by the addition of an equal volume of cryoguard-freezing extender. Spermatozoa were frozen according to the preset, porcine protocol (4 to 1°C in 1.3 min, to −10°C in 2.36 min, to –30°C in 3.54 min, and to –140°C in 14.56 min), using the IceCube 14S-B freezer. Frozen spermatozoa were stored at –196°C until analyses. Sperm motion characteristics were assessed before and after freezing (computer-assisted sperm analyzer), and their quality was evaluated post-thaw through plasma and acrosome membrane integrities (flow cytometry). Data were analysed (ANOVA-1), and P < 0.05 indicated significant difference. Motion analyses revealed dose-dependent and significantly increased proportions of motile (75 ± 5%, 77 ± 3%, and 84 ± 2%) and progressive (44 ± 4%, 49 ± 2%, and 64 ± 5%) spermatozoa, following exposure to MNP (0, 87.5, 175 μg, respectively; P < 0.05). The proportions of nanoselected spermatozoa with bent tails and distal droplets decreased significantly (P < 0.05). Freeze-thawing led to poor proportions of motile spermatozoa (<10%), regardless of the MNP concentrations. Control and nanoselected spermatozoa had similar post-thaw motility (P > 0.05). Flow cytometry analyses indicated comparable proportions of spermatozoa with intact membranes (P > 0.05). However, fluorescence intensities of staining dyes were decreased in nanoselected spermatozoa, indicating possible reduced sensitivity to further insults. We concluded that (1) the designed MNP allowed for high-throughput semen enrichment with highly motile spermatozoa, (2) nanoselection was more likely beneficial to semen with poor sperm motility (<70%), and (3) nanoselected spermatozoa may have greater robustness that did not show through cryotolerance. Our freeze-thawing protocol is under optimization for further analyses of nanoselected spermatozoa.

This research was supported by USDA-ARS Grant# 58-6402-3-018.