Mitochondrial bioenergetics analysis on SLC-selected boar spermatozoa during liquid storage

Semen preservation at 17 °C plays a pivotal role in the porcine breeding industry, though it presents challenges; not all ejaculates retain their quality during storage. Colloidal centrifugation has been developed to improve it since this method eliminates seminal plasma and microorganisms while simultaneously allowing the selection of spermatozoa based on gradient density centrifugation. This study aimed to evaluate whether two different colloid densities (1.026 g/ml – 20 %Porcicoll® and 1.104 g/ml – 80 % Porcicoll®) improve sperm bioenergetics, assessed using Agilent seahorse, along with key semen quality parameters such as viability, mitochondrial activity, oxidative stress and motility analyzed through flow cytometry and the CASA system over time. Fifteen ejaculates were divided into three groups: Control (CTR), 20 % Porcicoll® (P20), and 80 % Porcicoll® (P80). Sperm parameters were analyzed at days 0, 3, and 7 of storage. Results revealed a metabolic shift from mitochondrial oxidative phosphorylation to glycolysis during storage, with a 15 % decrease in ATP production by day 3. P80-treated sperm showed higher ATP production on day 0 compared to P20. Sperm quality parameters such as viability and mitochondrial activity decreased after colloidal centrifugation, possibly because of early capacitation; we also hypothesize that SLC could increase sensitivity to the high glucose levels in the medium. Although Porcicoll® effectively selected metabolically active sperm, the lack of seminal plasma and limitations in media composition negatively affected long-term sperm quality. Further research is necessary to optimize media formulations and assess fertility outcomes of selected sperm for enhanced sperm preservation techniques.