The protective effect of a 17 °C holding time on boar sperm plasma membrane fluidity after exposure to 5 °C

The holding time (HT) is the period during which an ejaculate, either in a raw or diluted state, is held at 17 °C before further processing for cold-storage. In boars, the HT positively influences select sperm quality parameters of semen cooled from 15 to 5 °C, a range in temperature during which plasma membrane remodeling occurs. Objective insight into the effect of HT on plasma membrane organization remains unknown. Therefore, the present work sought to elucidate if HT contributes to minimizing alterations in boar sperm plasma membrane fluidity at the initial step of the cooling process in a cryopreservation practice (holding at 5 °C) and in relation with select sperm quality parameters. Nineteen ejaculates from five boars were collected and processed according to different treatments: T1) Fresh diluted semen, 0 h at 17 °C; T2) Fresh diluted semen, 24 h at 17 °C (HT); T3) Sperm from T1 in a lactose-egg yolk (LEY) extender, 3 h at 5 °C; T4) Sperm from T2 in LEY, 3 h at 5 °C; T5) Sperm from T1 in LEY, 24 h at 5 °C; T6) Sperm from T2 in LEY, 24 h at 5 °C. Sperm motility was assessed using CASA, and sperm plasma membrane integrity and fluidity were evaluated by flow cytometry with dual labeling (M540/YO-PRO®-1). Results demonstrated that the lack of exposure to a HT (T5) results in reduced sample motility compared to those having a HT (T6), with sperm exposed to HT exhibiting less plasma membrane fluidity. Collectively, these results provide empirical evidence that incorporation of a HT in semen processing protects boar sperm against cold injury through maintenance of lipid architecture of the plasma membrane.