In vitro aging of boar spermatozoa: role of sperm proximity and seminal plasma

Abstract Background Knowledge on the effect of seminal plasma on sperm function in extended semen during in vitro storage is lacking. Objectives The aim was to examine the interactive role of sperm concentration and seminal plasma concentration on boar sperm function during in vitro aging. Material and methods Experiment 1: Twenty‐one boar ejaculates were aliquoted with Beltsville Thawing Solution into five semen doses containing between 32.5 and 8.5 × 10 6 sperm/mL. Experiment 2: Semen samples ( n = 8) containing 18 × 10 6 or 10 × 10 6 sperm/mL with their natural amount of seminal plasma and 10 × 10 6 sperm/mL substituted with autologous seminal plasma to the same concentration as in doses with 18 × 10 6 sperm/mL were prepared. Experiment 3: Four variants of semen doses containing 18 × 10 6 or 10 × 10 6 sperm/mL with either 10% or 0.5% (v/v) seminal plasma were used. Lipid peroxidation was assessed using Bodipy 581/591 in samples ( n = 8) with two different sperm concentrations. Semen was examined during 144‐h storage at 17 °C by computer‐assisted semen analysis and flow cytometry. Results Experiment 1: 3D kinematic patterns revealed a concentration‐ and time‐dependent loss of sperm kinematics in samples with < 23 × 10 6 sperm/mL ( p < 0.05). Percent viable spermatozoa with high mitochondria membrane potential were lower ( p < 0.05) in samples with < 15 × 10 6 sperm/mL. Experiment 2: Seminal plasma supplementation in samples with 10 × 10 6 sperm/mL did not restore the loss of sperm kinematics ( p > 0.05). Experiment 3: At 144 h, motility was lowest in samples containing 10 × 10 6 sperm/mL and 10% (v/v) seminal plasma ( p < 0.05). Sperm lipid peroxidation did not differ between samples with different sperm concentration. Conclusion Long‐term exposure to seminal plasma has a negative impact on in vitro‐aged boar spermatozoa. Reduced sperm‐to‐sperm proximity but not the reduction of seminal plasma limits sperm function in long‐term stored boar semen.