The Effect of Semen Washing and Soybean Lecithin Level on Motility and Viability of Ram Spermatozoa Stored at 5⁰c

The objectives of this reserarch were to study the effect of semen washing and soybean lecithinlevel on the motility and viability of ram spermatozoa stored at 5⁰C. Semen was collected from threemature rams by artificial vagina. Semen was evaluated by macroscopic and microscopic. Semen wasdivided into ten tubes, the semen of first five tubes was unwashed (W0) and diluted with extender 0%,1%, 2%, 3% and 4% soybean lecithin (L0, L1, L2, L3 and L4). Semen of another five tubes werewashed (W1) then each diluted with similar extender levels. The diluted semen samples were stored at5⁰C and the sperm motility and viability were evaluated each day. The interaction of the semen washingand soybean lecithin levels were no significant differences (P>0.05) but the single factor of soybeanlecithin level was significant differeNces (P<0.05) and the single factor of semen washing were nosignificant differences (P>0.05) on progressive motility and viability of ram sperm.The best extenderwas 3% soybean lecithin (L3) with the percentage of ram sperm progressive motility was 63.18 ± 3.61%and viability was 71.76±2.32%.


INTRODUCTION
The problems of cold shock in sperm preservation can be partly overcome by the use of protective agent in the extender. The popular protective agent used universally for sperm preservation or cryopreservation are glycerol and lecithin (Salamon and Maxwell, 1995). The main source of lecithin that has been long time used is the egg yolk, but the use of such materials as lecithin source to prevent the effects of cold shock has the risk on microorganisms contamination that jeopardize the spermatozoa and the female reproductive tract (Aku et al., 2007). These microorganisms are bacteria (Bousseau et al., 1998), especially Salmonella thyphimurium (Froning, 1998). Some other weakness of egg yolk as the protectant of spermatozoa are: (a) causing turbidity in the sperm dilution that lead to complicate the observation process under microscope when evaluating the spermatozoa quality (Aku et al., 2007), (b) only effective for the sperm preservation at temperature of 5⁰C so that it is not optimal in protecting spermatozoa from cooling effect under freezing point, and (c) at high concentrations it can decrease post thawing viability of ram spermatozoa (Fukui et al., 2008). Based on these considerations, it is necessary to develop alternative protectant that has a high quality than lecithin derived from egg yolk.
One of the potential protectant developed in protecting spermatozoa from detrimental preservation effect is soybean lecithin. This material is one of the vegetable lecithin that has most important phospholipids content to protect the spermatozoa membrane in preservation process. Naturally, the lecithin content in soybean is from 1.48 to 3.08%.
The main component of soybean lecithin is phospholipid composed of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositols and glycolipids (Shurtleff and Aoyagi, 2007). In the soybean lecithin was not found any microorganisms that can damage the spermatozoa or female reproductive tract (Bousseau et al., 1998).
The effectiveness of using soybean lecithin as component of the ram semen extender to protect spermatozoa on cold shock is hindered by some seminal plasma components that the bulbourethral glands produces. The bulbourethral glands secrete enzyme, called phospholipase A (Chemineau et al., 1991). Phospholipase A in the ram semen (Scott and Dawson, 1968;Roldan and Fragio, 1993) may catalyse the hydrolysis of lecithin in fatty acid and lysolecithin that are toxic for spermatozoa (Chemineau et al., 1991). Particularly in the buck semen, removal of plasma seminal by washing and centrifugation is routine procedure if diluted with extender containing egg yolk lecithin (Evans and Maxwell, 1987;Chemineau et al., 1991). In the ram semen, the effectiveness of removal of seminal plasma in this way before diluted with soybean lecithin was not exists its explanation. The accurate information on the effect of semen washing and soybean lecithin levels to the ram sperm quality is needed.

MATERIALS AND METHODS
Three mature rams were used as semen donors. Semen was collected by artificial vagina once every three days. The semen volume, semen colors, semen consistency, mass activity, sperm concentration, sperm morphology, sperm motility were measured. Only ejaculates with a concentration of greater than 250x10 7 sperm/mL, having >75% progressively motile sperm and <15% of the sperm with abnormal morphology were selected for this research.

Semen Preparation, Washing and Dilution
Fresh semen was equally divided into two tubes, both were unwashed (W0) and washed semen (W1).Each semen from both tubes of unwashed and washed semen was equally divided into five tubes, the five tubes of unwashed semen were immediately diluted with extender tested (L0, L1, L2, L3, and L4). The five tubes of washed semen were diluted after semen washing process. The semen were washed with the Krebs-Ringer phosphate glucose solution (Chemineau et al., 1991).
The percentage of motile and viable sperm was daily evaluated (24 hours) for five days of storage. The calculation of sperm motility based on the percentage of progressive motile sperm (Salmin, 2000) using formula: Sperm motility (%) = [(Total of sperm observed -Total of not progresive motile sperm)/(Total of sperm observed)] x 100% The criteria of not progressive motile sperm are moving backwards, vibrating in place, moving back, moving in circles and not moving at all or die.
The calculation of viable sperm was based on the percentage of live sperma through differential staining with eosin-negrosin stain (Bearden et al., 2004) using formula:

Data Analysis
The data were statistically analyzed for differences among the means by two way analysis of variance. The Duncan's Multiple Range Test was used to compare the treatment means using statistical software SPSS 17 version.

RESULTS AND DISCUSSION
Fresh semen (Table 1) used in this research were found eligible for further processing, especially for dilution and preservation due to its in the normal range recommended by some experts. Macroscopically, the normal standards of ram fresh semen is 0.5 to 2.0 ml in volume (Ax et al., 2000;, thin to thick creamy in consistency, milky-white or pale cream in color (Toelihere, 1985;Evans and Maxwell, 1987;Ax et al., 2000), 5.9 to 7.3 in pH (Bearden et al., 2004). Microscopically, the normal concentration ranges from 1500-3800 million sperm/mL, percentage of sperm motility and viability were 75.0 to 89.8% and 87.33 to 94.2%  and percentage of sperm abnormality were 8 to 10 % (Bearden et al., 2004). The sperm quality after semen washing (Table 2) was also still in good condition for being further processed. The success of semen washing process have been reported by many researchers with a variety of methods (Somfai et al.,2002;Councel et al., 2004;Lee et al., 2009;Marcus, 2010).
The interaction of semen washing and soybean lecithin levels in this research found no significant differeces (P>0.05) on the sperm progressive motility and viability (Table 3 and  Table 4) but the single factor of the soybean lecithin level was significantly (P<0.05) affects the motility and viability of the ram sperm for five days of storage at temperature 5⁰C. The semen washing treatment had no significant effect (P>0.05) on the sperm progressive motility and viability of ram for five days of storage. This phenomenon was indicated that ram semen diluted with soybean lecithin was not necessary being washed to remove the plasma seminal.
The use of soybean lecithin as extender did not give effect that can disrupt the function, motility and viability of ram spermatozoa. This was presumably because the phospholipase A enzyme in the plasma seminal of ram semen can tolerate soybean lecithin levels used in this research, so it did not catalyse the hydrolysis of soybean lecithin in fatty acids and lysolesitin that has caused coagulation of extender medium and toxic for spermatozoa. Most spermatozoa was still survival in motion and activity to maintain its viability in the extender medium enriched with soybean lecithin. This fact similar to the existence of egg yolk as a source of lecithin component in the ram semen extender that does not cause the spermatozoa death due to toxicity and coagulation of the extender medium. This phenomenon is supported by Ritar and Salamon (1982) that the viability of ram sperm was still not change when there is exist or no egg yolk in the ram semen extender. The opinion was also in line with several other researchers who use egg yolk as a source of lecithin component in extender medium of ram semen to against cold shock in preservation and cryopreservation (Herdiawan, 2004;Fukui et al, 2008;Yulnawati and Herdis, 2009). Unlike with the buck semen, the phospholipase A in its plasma seminal can catalyse the hydrolysis of egg yolk lecithin in fatty acids and lysolesitin which in large quantities was causes coagulation of extender medium and toxic for spermatozoa (Chemineau et al., 1991;. Although in the small levels, i.e. 2.5% egg yolk in the buck plasma seminal has most negatively affect on spermatozoa (Ritar and Salamon, 1982) so that it needs to be washed firstly (Evans and Maxwell, 1987;Chemineau et al., 1991). The ram semen diluted with egg yolk or other lecithin sources include soybean lecithin was not recommended for being washed. In contrast, the ram plasma seminal is often used by many researchers as substitution of the buck plasma seminal that has been removed to avoid toxicity and coagulation (Ritar and Salamon,1982;Souhoka et al., 2009;Ari and Daskin, 2010).
The positive effects of soybean lecithin in the ram semen extender appeared from the data trend of ram sperm motility and viability for five days of storage at temperature 5⁰C (Table 3 and  Table 4). This study indicated that 3% soybean lecithin level in extender was optimal level to increase the progressive motility (63.18 ± 3.61%) and viability (71.76±2.32%) of ram sperm for five days of storage at 5⁰C.
The high of progressive motility and viability of ram sperm at the 3% soybean lecithin levels (L3) was presumed because at the levels of the soybean lecithin are relatively effective to protect sperm membrane against cold shock at temperature 5⁰C. The motility and viability was maintained, mortality can be reduced, morphology undisturbed and sperm membrane remained intact (data not shown). At these levels, the spermatozoa avoided from bad influences of temperature refrigeration 5⁰C in the form of cold shock. The sperm are experiencing cold shock resulted in curled tail, swirling motion and backward motion so disturbing and decreasing its progressive motility (Evans and Maxwell, 1987). In addition, the cold shock also resulted in disruption of function and cell membrane structure which causes the sperma death (Souhoka et al., 2009;Rizal, 2006). In morphology, spermatozoa experiences cold shock was characterized by coiled tail or curled tail which became one of parameters of the sperm abnormality. The shape of sperm tail is like coiled Semen Washing and Soybean Lecithin on Motility of Ram Sperm (Salmin et al.) 247 or curled tail were categorized as secondary abnormalities (Partodihardjo, 1992). Most abnormal spermatozoa in the part of tail cannot motile (Bearden et al., 2004).

CONCLUSION
Based on this research results it was concluded that use of soybean lecithin as extender does not give effect that can disrupt the motility and viability of ram sperm. The ram semen diluted with soybean lecithin was not necessary washed to remove the plasma seminal. The suitable extender for the best quality of ram semen stored at 5⁰C was 3% soybean lecithin as protectant agent.