Effect of different levels of soybean lecithin and egg yolk on the motion parameters of goat spermatozoa after freezing – thawing

ackground: In recent years, several studies was performed about the effect of soy lecithin on sperm cryopreservation Including, goat(Jiménez-Rabadán, Ramón et al. 2012, Roof, Bowley et al. 2012, Salmani, Nabi et al. 2013, Salmani, Towhidi et al. 2014), ram (Sharafi, Forouzanfar et al. 2009, Forouzanfar, Sharafi et al. 2010, Valle, Gómez‐Durán et al. 2012), human (Reed, Ezeh et al. 2009) and cat (Vick, Bateman et al. 2010). Data between studies are different and various and optimal level of soy lecithin as a standard level is not known. Therefore, the aim of this study was to determine the optimal level of soy lecithin as a suitable alternative to egg yolk freezing goat semen. Sperm motility and motion parameters, the percent of normal sperm, plasma membrane integrity and apoptosis were assessed for evaluation of extenders containing different concentrations of soybean lecithin Methods: All chemical reagents were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated. For this study, six ejaculates from each goat were collected by artificial vagina twice a week during the breeding season from four mature Mahabadi bucks (3 and 4 yr of age) known to have good fertility. volume varying between 0.75 and 2 mL, sperm concentration of 3 × 109 sperm/mL, motile sperm percentage higher than 70%, and less than 10% abnormal sperm. To eliminate individual differences, semen samples from the four bucks were pooled. Each pooled sample was split into five equal aliquots and diluted with five extenders. The basic extender used in this study was composed of 30.7 g tris (Merck, Darmstadt, Germany), 12.6 g fructose (Merck) and 16.4 g citric acid (Merck). The osmolarity and pH were set at 420 mOsm and 7.2, respectively. Five different extenders were prepared by the addition of 1%, 2%, 3% and 4% soybean lecithin and extender containing 15% egg yolk as control treatment. Concentration of glycerol and the procedure for freezing was based on previous study (Salmani, Towhidi et al. 2014). The diluted semen samples were maintained at room temperature (∼25 ◦C) for 5 min and then equilibrated at 4 ◦C for 2.5 h. The cooled semen was loaded into 0.25-mL French straws at concentration of 1×109 sperm/ mL, according to the method described (Matsuoka, Imai et al. 2006), then sealed with polyvinyl alcohol powder. The straws were exposed to liquid nitrogen vapor, 4 cm above the liquid nitrogen for 12 min (Purdy 2006). Subsequently the straws were plunged into the liquid nitrogen for storage. After storage for 2 month, the frozen straws were thawed individually at 37 ˚C for 30 s in a water bath for microscopic evaluation. Evaluation of sperm after freezing-thawing Sperm motility was carried out according to Gil et al (Gil, Lundeheim et al. 2003). For this purpose, three straws from different freezing treatments were thawed at 37 8C for 10 sec and pooled in a test tube. Percentage of sperm motility was assessed using a computer-assisted sperm analysis system (CASA, Version12 IVOS, Hamilton-Thorne Biosciences, beverly, MA, USA). Semen samples were diluted 1:1 with tris buffer medium and incubated at 35 ˚C for 5 min. For evaluation, 10 μl drop of the sample was placed on a pre-warmed (37 ˚C) slide and covered with a 24 × 24-mm coverslip and a minimum of 200 sperm were counted under a phase contrast microscope at 37 ˚C and 100× magnification.Result: The results of this study showed that most of the post-thawed sperm motion parameters (ALH, VSL, VCL,) were not significantly different between EYT and L1% (P < 0.05). Although, LIN and STR was significantly in EYT In comparison with 1% (P < 0.05). In conclusion, the use of tris -based extender containing 1% Soybean lecithin for goat semen cryopreservation could, improve the motion parameters of goat spermConclusion: One of the most important factors in the first stage that showing the successful freezing is acceptable Sperm mobility followed the cryopreservation process. The results of total motility, ALH, VSL, VCL and LIN showed that, were not significantly (P < 0.05) different between EY and L1%. Our experiment shows that, optimum level of soybean lecithin for cryopreservation of goat sperm is 1%. Salmani et al. reported that the 1.5% of soy lecithin in diluent for freezing goat semen is most appropriate level (Salmani, Towhidi et al. 2014). The main reason for the decline sperm motility after freeze-thaw is biochemical and ultrastructural damage during the freezing process that can occur at different times (Aires, Hinsch et al. 2003). Freeze-thaw process results in changes in sperm morphology, which cause damage cell membranes, mitochondria and sperm acrosome. Causes the only a few percentage of sperms has the integrated membrane and normal activity of mitochondria followed by freeze-thaw which Resulting less active sperm count remains after freezing-thawing (Huopalahti, Anton et al. 2007). Sperm membranes release phospholipids into the surrounding medium during cold shock (Darin-Bennett, Poulos et al. 1973). Presumably phospholipids are implicated by interaction with the spermatozoa membranes and replacing some lipids, thus decreasing their phase transition temperature (Graham and Foote 1987)

کلیدواژه ها:

fertility ، Freezing ، Goat. Egg yolk ، Sperm ، Soybean Lecithin