Abstract

The cryopreservation procedure causes dramatic changes in boar sperm survival but it is yet unclear where and how the process affects spermatozoa. Cryopreservation damage appears partly associated with oxidative stress and reactive oxygen species (ROS) generation. The present study evaluates the effect that various steps of a conventional cycle of cryopreservation have on the intracellular production of ROS by boar spermatozoa (spz). Sperm-rich fractions collected from 2 mature boars (3 ejaculates per boar), cooled to 17°C, and kept for 16 h were cryopreserved following a standard freeze–thaw process with 0.5-mL plastic straws. The production of ROS was recorded in 5 steps of the cryopreservation process. These steps were as follows: step (1) after collection, when the fresh semen was extended (1:1, v/v) in Beltsville Thawing Solution (BTS, 205 mM glucose, 20.39 mM NaCl, 5.4 mM KCl, 15.01 mM NaHCO₃, and 3.35 mM EDTA); step (2) after cooling and storage for 16 h at 17°C; step (3) after centrifugation (2400g for 3 min) and re-extension of the pellet with lactose-egg yolk extender; step (4) at 5°C, after the addition of lactose-egg yolk-glycerol-Equex Stem Paste to 1 × 10⁹ spz mL; and step (5) immediately after thawing at 37°C for 20 s. For the ROS measurement, all samples were re-extended in BTS (3 × 10⁶ spz mL^-1) and incubated without (basal ROS level) or with ROS inducers (1 mM tert-butyl hydroperoxide) for 120 min at 37°C and 5% CO₂. Cells were simultaneously stained with 22,72-dichlorodihydrofluorescein diacetate (1 µM) to estimate the production of ROS, and propidium iodide (12 µM) to exclude dead sperm from the analysis. Samples were evaluated at 30 min and 120 min by flow cytometry (Coulter Epics XL; Coulter Corporation, Miami, FL, USA); further analyses of the parameters were done by FCSExpress software (DeNovo Software, Thornhill, Ontario, Canada). ROS production was expressed as the mean of the green intensity fluorescence units of the viable sperm population. Data from 3 replicates were analyzed as a split plot design using a mixed model ANOVA including cryopreservation step, boar, and incubation time as fixed effects and replicate as random effect. Results indicated that the basal ROS formation remained relatively low and constant (P = 0.95) through the cryopreservation process, without differences between boars (P = 0.559), although with a significant increase after 120 min of incubation (P < 0.001). However, the exposure to tert-butyl hydroperoxide significantly increased the intracellular ROS formation in all of the steps (P < 0.001), showing significant differences between them, and being especially raised at steps 3 and 4. In conclusion, the present study confirms that the basal intracellular ROS production during cryopreservation of boar sperm is low. Nevertheless, the susceptibility of those spermatozoa to external stresses vary through the cryopreservation process, especially after centrifugation and later extension at 17°C and after the slow cooling at 5°C.