Effect of seminal plasma and butylated hydroxytoluene (BHT) concentration on ram sperm freezability

The effect of removing seminal plasma (SP) by centrifugation before ram semen cryopreservation was assessed. Simultaneously, we studied the supplementation of different concentrations of BHT as antioxidant (0.6 mM, 2 mM and 5 mM) on the extender in order to find an optimal concentration in enhancing a successful ram sperm cryopreservation. Therefore, fresh ejaculates from 8 rams of approximately 2 years old were collected, immediately mixed in equal quantities and split into 2 aliquots. One aliquot was diluted (1:5) in Tris-citric acid-glucose (TCG) solution and washed twice by centrifugation at 600 × g for 10 min, while the other aliquot was kept unwashed. Afterwards, washed and unwashed sperm were re-suspended in one of different TCG-based media with 5% glycerol and 15% powdered egg yolk (PEY) supplemented or not with BHT and/or SP according to the treatments: 1) washed sperm; 2) washed sperm + 0.6 mM BHT; 3) washed sperm + 2 mM BHT; 4) washed sperm + 5 mM BHT; 5) washed sperm + 0.5 mL of SP; 6) washed sperm + 5 mM BHT + 0.5 mL of SP; 7) unwashed sperm and 8) unwashed sperm + 5 mM BHT. Afterwards, sperm samples were refrigerated at 5° C for 4 h before being packed in 0.25 mL straws and frozen in nitrogen liquid vapors. After thawing, plasma and acrosomal membrane integrity and mitochondrial activity were determined by flow cytometry while sperm motility and morphometry by CASA (mean ± SEM, n = 6). Our results showed that seminal plasma removal enhanced sperm cryosurvival and the addition of BHT (5.0 mM) improved most of the quality parameters of thawed washed and unwashed sperm, irrespective of the presence of SP in the extenders.