15 EFFECT OF BULL EPIDIDYMIS STORAGE CONDITIONS ON CRYOPRESERVED EPIDIDYMAL SPERM IN VITRO FERTILITY AND LIPID PEROXIDATION STATUS

Abstract

Although cryopreservation of epididymal sperm has been studied extensively in several species, some factors that could negatively influence its quality are still unknown, such as the storage conditions of the epididymides prior to sperm collection. Studies indicate that the lower the storage temperature, the better the sperm quality after collection (Kaabi et al. 2003 Theriogenology 60, 1249–1259). An additional factor is lipid peroxidation in which sperm membrane resistance against reactive oxygen species (ROS) attacks is an important component. The objective of this experiment was to study whether the epididymis storage temperature following slaughter, as well as the intrinsic resistance against oxidative stress, affects the fertilizing capacity of cryopreserved epididymal bull sperm in vitro. Twelve epididymides (6 bulls) were collected after slaughter and divided into 2 groups, (stored at either 4 or 37°C for 2), after which semen was collected from the caudae epididymides and cryopreserved. Subsequently, one aliquot of the frozen–thawed semen samples was subjected to induced lipid peroxidation with ferrous sulfate and ascorbate (37°C; 2 h), after which tiobarbituric acid-reactive substances (TBARS), as an index of lipid peroxidation, were measured according to a method previously described (Beorlegui et al. 1997 Andrologia 29, 37–42). A second aliquot was used for in vitro fertilization in a routine IVF–IVC setup in duplicate (24-h maturation, SOF culture medium in 5% CO₂, 5% O₂, and 90% N₂). In vitro embryo production results at Day 7 and TBARS levels were statistically analyzed using SAS (SAS Institute, Inc., Cary, NC, USA). No influence of storage temperature was observed at either TBARS level (4°C: 943.6 ± 173.4; 37°C: 751.4 ± 136.2 ng of TBARS/10⁸ spermatozoa; P = 0.3) or on blastocyst rates (4°C: 23.0 ± 2.8; 37°C: 18.7 ± 3.6% of blastocysts; P = 0.2). However, the percentage of hatched blastocysts tend to be higher for epididymides stored at 4°C when compared to those stored at 37°C (6.4 ± 1.8 and 2.3 ± 0.9, respectively; P = 0.06). In addition, a negative correlation was found between TBARS concentrations and blastocyst rates (R = –0.57; P < 0.05). Compared to fresh samples collected from epididymides under the same conditions (unpublished data), levels of TBARS were two- to threefold higher for the cryopreserved sperm, indicating that lipid peroxidation appears to play a role in the decrease in quality of cryopreserved epididymal sperm. In conclusion, temperature during the epididymides short-term storage prior to sperm cryopreservation did not seem to influence the sperms' in vitro fertilizing capacity. On the other hand, an alternative to improve cryopreserved epididymal sperm in vitro fertility (or fertilizing capacity) could be the addition of antioxidants to semen extenders. Further studies are necessary to confirm this hypothesis.