Effect of soybean Lecithin on quality, lipid peroxidation, antioxidant enzyme activity and acrosome membrane integrity of ram sperm after freeze-thawing

Introduction: The use of cryopreserved semen in artificial insemination (AI) has numerous advantages for the animal husbandry, especially when used in breeding programs. Long-term storage of sperm in liquid nitrogen is a valuable technique for genetic resources preservation (Mazur et al., 2008). Animals are transported frequently and widely around the world, and it is necessary to create and maintain suitable conditions during animal transportation (Salamon and Maxwell, 2000). However, the costs, the risks of escape, and the potential for accidental death of animals are unavoidable. On the other hand, cryopreserved sperm can be transported in liquid nitrogen at a markedly lower cost and without the risk of escape or death of animals (Aires et al., 2003).Numerous investigations have been performed to improve the protocols for cryopreservation of sperm. The extenders used for semen cryopreservation protect the sperm against thermal shock, preserving both motility and fertility by promoting the stabilization of the plasma membrane, and providing energy substrates (Amirat et al., 2004). These attributes reduce the deleterious effects of changes in the pH and osmolarity, prevent the growth of bacteria, and protect the sperm cells from the damage caused by refrigeration, freezing, and thawing. For sperm cryopreservation, protocols using egg yolk have been successfully established and applied in various animal species (Aisen et al., 2002). However, the use of egg yolk in cryopreservation has been questioned, because of certain potential negative aspects. Egg yolk contains a wide variety of compounds that are both beneficial and also potentially harmful to sperm. Additionally, egg yolk introduces a risk of contamination by microbes that can subsequently produce endotoxins; and so, the egg yolk and sperm are subjected to quarantine inspection in the case of import or export. Therefore, a replacement for egg yolk with a well-defined chemical composition would be very advantageous. Lecithin, also known as phosphatidylcholine, is a component of egg yolk and is known to prevent cold shock during the freezing and thawing process in sperm cryopreservation. Soybean lecithin in particular has already been used as a replacement for egg yolk in sperm cryopreservation in various animals. Its non-animal origin and minimal sanitary risks make it preferable for this application. Soybean Lecithin can be used as an extender in the cryopreservation process (Akhter et al., 2012).The aim of this study was to evaluate the effects of Soybean Lecithin as extender on the ram semen quality after freeze-thawing.
Material and methods: Semen was collected from the indigenous Ghezel rams. Twenty five ejaculates were collected throughout the entire study, with semen being collected twice a week (every Sunday and Tuesday) from each ram, using the artificial vagina. Ejaculates were collected in graduated test tubes, placed in a thermo flask at 34°C, and transported to the laboratory for evaluation within 1h interval. The raw or fresh undiluted semen was then microscopically evaluated for volume, concentration, and sperm motility. The sperm concentration was determined with the aid of a neubauer lam. A Computer Assisted Sperm Analysis (CASA) system was used to evaluate the different sperm motility characteristics. All data were analyzed using the statistical procedure of SAS (version 9.2). The analysis of variance (ANOVA) was used to test for significant differences between treatments. Characterization of the Ghezel ram sperm viability (percentage live/dead) of the semen samples was determined using an eosin/nigrosin stain (50μl eosin/nigrosin and 5μl semen). The volume of the ram ejaculates ranged between 0.75 and 1.5mL. The sperm concentration recorded in this study ranged between 0.9 and 1.3 × 10⁹ sperm/mL. The effect of different soybean lecithin levels on the Ghezel ram semen characteristics following cryopreservation was evaluated. After initial evaluation of the ejaculates, all ram ejaculates were pooled and semen sample was then divided into three portions; then, semen samples were diluted with soybean lecithin (1, 1.5 and 2 %) citrate extender and cooled over a period of 2h to 5°C. The semen samples were equilibrated for 2h and then loaded into 0.25mL semen straws. The straws were frozen in liquid nitrogen (LN2) vapor, then semen straws were plunged into the LN2 (-196°C). The semen straws were thawed 30 days later, in a water bath (37°C) for 30 seconds. The sperm characteristics included motility and motion parameters, viability, plasma membrane and acrosome integrity, sperm abnormality, and lipid peroxidation were evaluated. Motility and velocity were microscopically evaluated using the Sperm Class Analyzer® (CASA) system.
Results and discussion: This study demonstrated that soybean lecithin (1.5%) containing 7% glycerol can be used to cryopreserve Ghezel ram semen effectively, based on the sperm motility characteristics. The low sperm motility results recorded when semen was cryopreserved in soybean lecithin (2%). The results showed that total motility and some sperm motion parameters (VSL, VCL, VAP and ALH) were significantly higher in 1.5% lecithin soybean treatment group (P <0.05). In terms of survival, the percentage of abnormal sperm, plasma membrane integrity, and lipid peroxidation were not significant between treatments. Acrosome membrane integrity of 1.5% soybean lecithin treatment group was higher than other treatments (p < 0.05).