307 EFFECT OF BULL EPIDIDYMIS STORAGE CONDITIONS ON SPERM RESISTANCE AGAINST LIPID PEROXIDATION AND SUBSEQUENT IN VITRO EMBRYO PRODUCTION

Abstract

Sperm recovery from the cauda epididymis can be a usefull tool in case of unexpected death of genetic high-value animals or endangered species or when the collection of sperm by other means becomes impossible. Studies indicate that the lower the temperature of epididymis storage, the better the sperm quality after collection (Kaabi et al. 2003 Theriogeneology 60, 1249-1259). One of the main factors that can negatively affect sperm viability during storage is lipid peroxidation, where sperm membrane resistance against reactive oxygen species (ROS) attacks is an important factor. The objective of this experiment was to study whether the temperature of epididymis storage following slaughter would have an influence on the membrane's resistance against lipid peroxidation and on the sperm cell's fertilizing capacity. Sixteen epididymides (from eight bulls) were collected after slaughter and divided into two groups, one stored at 4°C and the other at 37°C for 2 h, after which semen was collected from the caudae epididymides. Sperm concentration was measured and an aliquot containing 10⁸ sperm cells was submitted to induced lipid peroxidation with ferrous sulfate and ascorbate (37°C; 2 h). Subsequently, thiobarbituric 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 of the sample was used for fertilization in a routine IVF-IVC set up in duplicate (24-h maturation, SOF culture medium in 5% CO₂, 5% O₂, and 90% N₂). In vitro embryo production and level of TBARS were statistically analyzed using SAS (SAS Institute, Inc., Cary, NC, USA). TBARS levels were transformed to logarithm form in order to obey the residue normality being analyzed using PROC GLM. The percentage of blastocysts was analyzed using the Wilcoxon test. When compared to the samples stored at 4°C, semen of caudae epididymides stored at 37°C showed higher levels of TBARS and lower mean blastocyst rates (324.7 ± 59.6 and 36.6 ± 1.6 vs. 466.9 ± 67.9 ng of TBARS/10⁸ spermatozoa and 28.8 ± 2.9%, respectively; P < 0.05). A negative correlation was found between TBARS and blastocyst rates (R = -0.43). The lower quality of sperm collected from epididymides maintained at higher temperatures may be related to a decrease in sperm resistance against lipid peroxidation which would further impair sperm fertilizing capacity. However, further studies are necessary in order to study the effect of temperature on the sperm membrane lipid profile, because the content of polyunsaturated fatty acids may be affected by temperature; this is an important factor relative to sperm membrane lipid peroxidation susceptibility (Ollero et al. 2000 Mol. Reprod. Dev. 55, 326-334). Another important factor is the epididymal environment because interactions between the sperm membrane and its surroundings can play an important role on the membrane's antioxidant protection.