title: Removing seminal plasma improves bovine sperm sex-sorting.
abstract: Bull ejaculates with sperm concentrations of less than 1 billion sperm sort poorly for sex chromosomes, but whether this is because of the sperm concentration or the concomitant seminal plasma content has not been elucidated. Experiments were conducted to determine why ejaculates with lower sperm concentrations sort poorly and develop a protocol to increase sorting efficiency. In Experiment I, spermatozoa at 160 or 240 × 106sperm/mL were stained at 49, 65 or 81 μm Hoechst 33342 with 0 or 10% seminal plasma and then sex-sorted. In Experiment II, seminal plasma was adjusted to create samples with sperm concentrations of 0.7, 1.4 and 2.1 × 109sperm/mL, prior to sex-sorting. In Experiment III, spermatozoa were diluted to 0.7, 1.4 and 2.1 × 109sperm/mL using TALP containing 0 or 10% seminal plasma prior to sex-sorting and cryopreservation. In Experiment I, the optimal staining combination was 160 × 106sperm/mL stained with 65 μm Hoechst 33342 and no seminal plasma. In Experiment II, the percentages of membrane-impaired sperm were lower for sample concentrations of 2.1 × 109sperm/mL (15%) than for samples at 1.4 × 109(17%) or 0.7 × 109sperm/mL (18%; p < 0.01). The X sort rate was slower for samples stored at 0.7 × 109sperm/mL (3.45 × 103sperm/sec) than for samples stored at 1.4 × 109and 2.1 × 109sperm/mL (3.85 and 3.94 × 103sperm/sec, respectively; p < 0.05). In Experiment III, samples containing 0% seminal plasma had higher percentages of live-oriented cells (54 vs. 50%; p < 0.05), fewer dead sperm (19 vs. 22%; p < 0.01) and higher post-thaw motility (41 vs. 35%; p < 0.05) than samples containing 10% seminal plasma. Ejaculates with high sperm concentrations result in superior sorting because these samples have less seminal plasma during staining than ejaculates with lower initial sperm concentrations as all samples are diluted to 160 × 106sperm/mL for staining. Therefore, sorting efficiency appears to be affected by seminal plasma concentration, not by the original sperm concentration.
authors: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, USA.
authors: C B
authors: CB
authors: Steinhauser
authors: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, USA.
authors: J K
authors: JK
authors: Graham
authors: Sexing Technologies, Navasota, TX, USA.
authors: R W
authors: RW
authors: Lenz
authors: Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, CO, USA.
authors: G E
authors: GE
authors: Seidel
mesh_terms: D000818:Animals; D002417:Cattle; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D008297:Male; D012661:Semen / Q000166:cytology*; D012662:Semen Preservation / Q000379:methods*; D013076:Sperm Count; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology*
keywords: bull sperm; seminal plasma; sex-sorting