In brief: Evaluation of sperm samples with similar motility after thawing has limited value to identify differences in sperm bioenergetic capacity. Maintaining sperm for 24 hr at room temperature is sufficient to detect bioenergetic and kinematics divergences.
Abstract: Sperm transport through the female reproductive tract requires energy for motility and fertilization. Sperm kinematic assessment is conducted as an industry standard to estimate semen quality prior to bovine insemination. However, individual samples with similar post-thaw motility result in different pregnancy outcomes, suggesting that differences in bioenergetics may be important for sperm function. Thus, characterization of bioenergetic and kinematic parameters of sperm over time may reveal novel metabolic requirements for sperm function. Post-thawed sperm from five samples of individual (A, B, C) and pooled bulls (AB, AC) were assessed at 0 and 24 h after thawing. Sperm were evaluated for kinematics via computer-assisted sperm analyses and bioenergetic profiles using a Seahorse Analyzer for basal respiration (BR), mitochondrial stress test (MST), and energy map (EM). Motility was nearly identical among samples after thawing and no differences in bioenergetics were detected. However, after 24 h of sperm storage, pooled sperm samples (AC) presented with higher BR and proton leakage compared to other samples. Sperm kinematic variability among samples was higher after 24 h, suggesting difference in sperm quality may manifest over time. Despite a reduction in motility and mitochondrial membrane potential, BR was higher at 24 h compared to 0 h for nearly all samples. A metabolic divergence between samples was detected by EM, indicating a shift in bioenergetic profiles over time that was undetected after thawing. These new bioenergetic profiles elucidate a novel dynamic plasticity of sperm metabolism over time while suggesting an influence of heterospermic interactions for further investigation.