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 × 10⁶ sperm/mL. Experiment 2: Semen samples (n = 8) containing 18 × 10⁶ or 10 × 10⁶ sperm/mL with their natural amount of seminal plasma and 10 × 10⁶ sperm/mL substituted with autologous seminal plasma to the same concentration as in doses with 18 × 10⁶ sperm/mL were prepared. Experiment 3: Four variants of semen doses containing 18 × 10⁶ or 10 × 10⁶ 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 × 10⁶ sperm/mL (p < 0.05). Percent viable spermatozoa with high mitochondria membrane potential were lower (p < 0.05) in samples with < 15 × 10⁶ sperm/mL. Experiment 2: Seminal plasma supplementation in samples with 10 × 10⁶ 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 × 10⁶ 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.

Keywords: boar seminal plasma; cell-to-cell interaction; semen preservation; sperm aging; spermatozoa.