The Addition of Osteopontin for Freezing Epididymal Bovine Sperm Improves Post‐Thaw Motility, Morphology and Membrane Functionality

Cryopreservation of epididymal sperm is an important tool for preserving the germplasm of animals with high genetic value after death or due to clinical conditions. Nonetheless, its implementation remains challenging, as sperm are more susceptible to freezing and thawing. Osteopontin is a protein that has been linked to high fertilisation rates and antioxidant properties. The objective of this study was to evaluate the effect of adding osteopontin to the freezing extender on the post-thaw quality of bovine epididymal sperm. Sperm was collected from the epididymis cauda from 13 bovine testis-epididymal complexes from Bos indicus bulls. Each sample was supplemented with osteopontin at 0.1, 1.0 and 10 μg/mL, and a control treatment without osteopontin. The spermatozoa were extended and frozen in nitrogen vapour, then evaluated after thawing for motility, kinematics, morphology and plasma membrane integrity. Compared to the control treatment, the addition of osteopontin at 0.1, 1.0 and 10 μg/mL increased the total and progressive motility of frozen-thawed epididymal spermatozoa, with no differences among these three treatments. The use of osteopontin at 10 μg/mL increased the beat cross frequency (BCF) of spermatozoa; however, no other differences were found in the other kinematic parameters of thawed epididymal semen. An increase in the proportion of morphologically normal sperm was observed when the three concentrations of osteopontin were added to the freezing extender (0.1, 1.0 and 10 μg/mL). Similarly, all three concentrations of osteopontin improved the functional integrity of the plasma membrane of thawed epididymal sperm, regardless of the concentration used. It is concluded that adding 0.1 μM osteopontin to the freezing medium is enough to improve the quality of the frozen-thawed bovine epididymal spermatozoa, in an equivalent manner to the use of higher concentrations of this protein.