Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

Abstract

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 °C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 °C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples ( P<0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5±3.39%; 1.4±0.16 FPM versus 48.6±4.02%, 2.6±0.16 FPM; P<0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0±19.50 spermatozoa) and distance (56.3±5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5±2.77%) and FPM rating (2.8±0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0±3.67 spermatozoa, 21.6±3.05 mm, 12.2±1.31% and 1.2±0.10 FPM, respectively; P<0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0±1.2 mm; FT-GRADIENT: 57.2±0.76 mm; FT-CONTROL: 51.7±0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.