Seminal Plasma Proteins Prevent Detrimental Effects of Ram Sperm Cryopreservation and Enhance the Protective Effect of Lecithin

Abstract

Background: One of the main drawbacks of refrigerated and frozen-thawed semen is lipid peroxidation and the subsequent generation of reactive oxygen species from cellular metabolism. In this study, we tested the hypothesis that the use of antioxidant compounds improves the frozen-thawed sperm quality. We analysed the effect of different antioxidants, or antioxidant-like additives, upon the processes involved in cooling and freeze-thawing ram semen. Methods: Sperm motility, plasma membrane integrity and stability, and mitochondrial membrane potential (MMP) were tested immediately after thawing and following incubation for 3 h and 6 h at 37°C. Results: The addition of oleic/linoleic acid did not improve sperm viability, although it resulted in increased motility after refrigeration and rewarming, similar to that found with pyruvic acid. In frozen-thawed samples, the effect of 75 mM ascorbic acid was beneficial and improved viability, membrane stability, MMP and motility. Pyruvic acid, melatonin, pinoline and N-acetyl cysteine, or the combination of certain antioxidants such as oleic/linoleic acids with tocopherol, lipoic and ascorbic acids, melatonin and pinoline, N-acetyl cysteine and GSH did not significantly improve the frozenthawed sperm quality. Thawed samples supplemented with lecithin scored higher (p<0.001) membrane integrity and stability and MMP values than controls. However, the alterations induced in the inner mitochondrial membrane resulted in a very low proportion of functional mitochondria in a time-dependent manner after thawing. These detrimental effects were prevented by seminal plasma proteins, which enhanced the protective effect of lecithin. Conclusion: Seminal plasma proteins strengthened the cryoprotective ability of lecithin and not only were sperm viability and membrane stability well maintained, but mitochondrial functionality was preserved. General significance: Lecithin together with seminal plasma proteins may be used as cryoprotectants for ram semen.