This study was conducted to determine the optimum level of glycerol and cholesterol-loaded cyclodextrin (CLC) in a Tris-based diluent for cryopreservation of ram spermatozoa. Ram semen was treated with 0, 1.5, 3 or 4.5 mg CLC/120 × 10(6)  cells in Tris-based diluents containing 3, 5 or 7% glycerol in a factorial arrangement 3 × 4 and frozen in liquid nitrogen vapour. Sperm motility, viability (eosin-nigrosin staining) and functional membrane integrity (hypo-osmotic swelling test) were assessed immediately after thawing (0 h) and subsequently after 3 and 6 h at 37°C. There was an interaction between CLC and glycerol on the functional membrane integrity (p < 0.05). In the presence of 3% glycerol, the highest functional membrane integrity (32.2%) was found in the spermatozoa treated with 1.5 mg CLC/120 × 10(6)  sperm. Post-thaw sperm motility was highest in 1.5 mg CLC immediately after thawing (40.5%) and after 3-h (30.6%) incubation at 37°C (p < 0.05). Viability of spermatozoa was higher in all CLC treatments than in the untreated samples, and it was highest (33.9%) in the spermatozoa treated with 1.5 mg CLC (p < 0.05). These data indicate that the addition of cholesterol to sperm membranes by 1.5 mg CLC/120 × 10(6)  cells may allow the use of a lower concentration of glycerol (3%), which is sufficient to mitigate the detrimental effects of freezing and thawing.

No abstract

This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non-cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non-cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non-cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non-cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.

This experiment was designed to investigate leptin receptors (Ob-R and Ob-Rb) mRNA in ram epididymal spermatozoa by RT-PCR. Ten testes were obtained from abattoir and epididymal spermatozoa recovery was performed. To purify spermatozoa, motile sperm were isolated by the swim-up procedure. Total RNA was isolated from epididymal spermatozoa and placental cotyledon, as a positive control, and then they were purified. Specific bands (98 and 308 bp) for Ob-R and Ob-Rb were detected after RT-PCR in both epididymal spermatozoa and placental cotyledon. We may conclude that Ob-R and Ob-Rb mRNA are present in ram epididymal spermatozoa and leptin perhaps exerts physiological effects, as already demonstrated in human.