Effects of hyaluronic acid on sperm parameters, mitochondrial function and apoptosis of spermatozoa in Simmental bulls with good and poor freezing ability

Abstract Bulls with varying freezability exhibit substantial variation in semen characteristics after cryopreservation. Sperm freezability is positively correlated with membrane cholesterol content, membrane integrity, mitochondrial activity and antioxidant content. The purpose of this study was to determine the optimal concentration of hyaluronic acid (HA) in bull sperm with different cryotolerances. Simmental bulls ( n = 10) semen samples were taken and categorized based on their progressive motility (PM) after freeze‐thawing: Group I, consisting of bulls ( n = 5) with progressive sperm motility ≥45%, was considered good freezability ejaculates (GF), and Group II, including bulls ( n = 5) with progressive sperm motility ≤30%, was considered poor freezability ejaculates (PF) bulls. Semen samples were diluted with a Tris‐egg‐yolk‐glycerol (TEYG) extender containing various concentrations of HA: without HA (control), 1 mM HA, 2 mM HA and 4 mM HA. After the freeze‐thaw process, sperm kinematics, plasma membrane and acrosome integrity, mitochondrial activity and apoptotic status were evaluated. The addition of 1 mM HA to the diluent of bulls with GF increased PM and linearity (LIN) compared to the control group ( p < 0.05). Normal morphology was improved after thawing in the samples treated with 1 and 2 mM HA in the GF and PF bulls respectively. The membrane and acrosome integrity of GF bulls treated with 1 mM HA was significantly ( p < 0.05) greater than that of the control groups. Adding 1 mM HA to the extender of bulls with GF and PF improved the proportion of viable cells compared with the highest concentration (4 mM) of HA. The mitochondrial activity of PF bulls treated with 1 and 2 mM HA was significantly ( p < 0.05) greater than that of the controls and 4 mM HA. Finally, it can be concluded that adding low doses of HA (1 mM) to the TEYG extender of GF and PF bulls ameliorated the post‐thaw semen quality.