The aim of the study was to ascertain effects of different concentrations of relaxin added to
extender medium during the pre-freezing incubation periods on quality variables of stallion
frozen-thawed spermatozoa. Semen samples collected from three stallions were filtered, diluted
with skim milk, and centrifuged at 600g for 10 min. Sperm pellets were suspended in BotuCrio
freezing medium to a final concentration of 50 × 106 sperm/mL. The diluted semen was divided
into five experimental groups supplemented with 0 (control), 12.5, 25, 50, or 100 ng/mL of relaxin.
The semen samples were transferred into 0.5 mL straws, equilibrated at 5 °C for 30 min, and
placed in liquid nitrogen (LN2) vapour for 15 min before being plunged into LN2. After thawing,
sperm samples were evaluated for motility and velocity variables, mitochondrial membrane potential,
apoptosis, and plasma membrane and DNA integrities. For sperm motility variables, there
were dose- and time-dependent effects, with the largest values recorded when 12.5 and 25 ng/mL
relaxin were used for 0–120 min of incubation. Furthermore, at all of the concentrations at which
there were evaluations, relaxin additions to semen diluent led to a marked improvement in sperm
mitochondrial membrane potential and a lesser percentage of apoptotic cells compared to the control
group. Plasma membranes and DNA integrities were not affected by relaxin supplementations
to the diluent. In conclusion, supplementation of relaxin in extender before semen cryopreservation,
especially at 12.5 and 25 ng/mL, had a positive effect on the sperm quality variables.