Comparison of Commercial and noncommercial Medium on Sperm Motility after Freezing

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1 International Research Journal of Applied and Basic Sciences 2013 Available online at ISSN X / Vol, 7 (12): Science Explorer Publications Comparison of Commercial and noncommercial Medium on Sperm Motility after Freezing Azita Azarpoor 1, A.T.Sarami 2, MaarefatGhaffari Novin 3 1. Master of Azad University Science and Research, Department of Molecular Cell Biology 2.gynaecologist,subspecialty in infertility, ivf a laparoscopic surgery,sarem cell research, centersaremwomenshospital,tehran, Iran 3. MD,Phd Corresponding Author:AzitaAzarpoor ABSTRACT:In general, human sperm is preserved in temperature range under -20 C. Currently, survival rate of the post-thaw sperm is around 50-60%. Although sperm cryopreservation medium including proteins and lipids derived from animals, for example egg yolk as commonly used, the components of proteins and lipids derived from animals are not clear. Therefore, synthetic and plant derived medium was used for the study. In the present study, post-thaw motility frozen with medium including long-chain alcohol and lipids was examined to develop cryopreservation medium for human sperm.during post-cool evaluation of the various extenders no differences were found in the motility between extenders. This observation was anticipated, as there are canine semen extenders commercially available void of egg yolk. An unexpected result was the significant 11% decrease of normal morphology in the 0% egg yolk concentration extender after 3 hours of cooling at 5 C with no significant change in the remaining extenders. Keywords:Comparison, Sperm Motility, Freezing. INTRODUCTION In general, human sperm is preserved in temperature range under -20 C. Currently, survival rate of the post-thaw sperm is around 50-60%. Although sperm cryopreservation medium including proteins and lipids derived from animals, for example egg yolk as a cryoprotectant, is commonly used, the components of proteins and lipids derived from animals are not clear. Therefore, synthetic and plant derived medium was used for the study. In the present study, post-thaw motility frozen with medium including long-chain alcohol and lipids was examined to develop cryopreservation medium for human sperm. Over the years researchers have tested and proposed numerous freezing extenders of various compositions in attempt to improve the quality and use of frozen stallion semen. Comparisons among stallion freezing extenders are documented poorly. It has also been reported that the most effective semen extender for one stallion is not necessarily the most effective for another. In 1984, Palmer indicated that freezing horse semen could use the same milk diluents (used for diluting semen immediately after collection) with the addition of 2% egg yolk and 2.5% glycerol [3]. The composition of this extender resulted in the formation of the milk based Modified French formula freezing extender (MFR5) that is used commercially today for freezing equine spermatozoa. The Lactose EDTA (LE) freezing extender is another commercially used equine freezing extender that is egg-yolk based. The composition of this extender developed by Martin et al. in 1979 was an experimentally developed extender formulated by combining two extenders; lactose and EDTA. Lactose EDTA resulted in higher post-thaw progressive motility (53.4%) when compared to the control (42.3%) which was composed of lactose, egg yolk, and glycerine. MATERIALS AND METHODS Semen samples were obtained from 45 (n=45) patients. All samples had normal concentrations ( 20 X 106 ml). Routine semen analysis was performed under light microscopy to determine the % motility and % forward progressive motile spermatozoa of the samples. Th e semen from each patient was divided into four equal aliquots and cryopreserved in each of the sperm cryopreservation media according to product instructions. After thawing, the motility and % forward progression for each sample were determined, and the recovery compared to the pre-freeze values was calculated. Sperm motility Twenty microlitres of each sperm sample was placed in a prewarmed (38 8C) Makler counting chambe r (Sefi Medical Instruments, Haifa) and immediately analysed with a CASA instrument (SM -CMA; MTM Medical Technologies, Montreux, Switzerland) on eight predetermined optical fields around the central reticulum of the chamber. A minimum number of 200 spermatozoa were accounted for per sample. The percentage of progressively motile spermatozoa and the straight linear velocity (VSL; mm/s) were determined. Sperm motility was assessed in a microscope equipped with 38 8C microscope stage and phase contrast optics (200!; Optiphot-2; Nikon, Japan), using the CASAinstrument (Microptic, SL, 2002).

2 Statistical analysis For the evaluation of sperm integrityandmotility, statistical comparisons of samples were performed by multi-anova (MANOVA). Student s t-test was used to compare least square means, and a general linear model was included to assess the differences among the different classes. The level of significance was set at P<0.05. Effect of cryopreservation on spermatozoa Spermatozoa continuously change and develop from their origins as somatic cells until their destination as highly specialized cells capable of fertilization. They have basically three functional regions comprising a head that contain the condensed nuclear material, a mid piece serving as a powerhouse and a tail which is the propulsive region. Subsequent maturation occur within the epididymis, followed by further development induced first by contact with seminal plasma and then by the secretions of the female tract (Varner and Johnson, 2007). The fi nal stages of spermatozoon development are induced by the immediate environment of the oocyte and its zona. In the process, most of the organelles are lost together with the cytoplasm, and the spermatozoal chromatin is remodeled. This specialization, though, is achieved at a cost, reducing the spermatozoon s ability to repair itself leading to a greater susceptibility to environmental change. Hence, even under ideal conditions, it is inevitable that some damage will occur to spermatozoa during the freezing process (Andrabi, 2007). The structural changes produced in the post thaw sperm cells membrane are primarily linked to altered abilities for energy sourcing. This would later on influence both cellular metabolism and other sperm functions such as motility (Cerolini et al, 2001; Dziekonska et al, 2009; Gillan et al, 2004). A spermatozoon is one of the smallest cells in the body specifically designed for propagation of genetic material achieved through fertilization. Like many other cells in the body, it requires a constant supply of energy for maintenance of cellular order and functions needed for survival and accomplishment of its task. This energy requirement increases significantly with the onset of activated motility, and becomes even more pronounced when hyperactivated motility is initiated (Granish and Suarez, 2002; Varner a nd Johnson, 2007). Plenty of exogenously derived nutrients are required by the spermatozoa to gain the strength needed for the long journey from the epididymis to the ovum in the female reproductive tract. These nutrients are metabolized intracellularly, resulting in the release of useable energy available for cellular processes primarily in the form of ATP. Like many metabolically active body cells, spermatozoa possess the metabolic machinery required for glycolysis, the citric acid cycle, and oxidative physphorylation. ATP for spermatozoa is mainly derived either by glycolysis in the cytoplasm or throughoxidative phosphorylation in the mitochondria (Dziekonska et al, 2009; Januskauskas, and Zillinskas, 2002). The relative contributions of the two processes to ATP generation are as yet unclear. A gradual reduction in the metabolic activity of spermatozoa during storage at cold shock temperature could limit the production of detrimental by-products, which might compromise sperm function but metabolic activity altered in this way does also influence essential sperm functions such as motility. Among the different alterations of activity of the intracellular enzymes, glucose-6-phosphate-dehydrogenase is the first enzyme which leaves the cell when the cellular membrane is damaged during cold shock. Generally, the intracellular concentration of ATP is decreased or lost and the AMP/ADP-rate is increased by the cryopreservation. Spermatozoal motility, considered to be one of the most frequently used characteristics for evaluating the fertility potential of ejaculated spermatozoa, is known to be dependent on mitochondrial function. The ATP generated by oxidative phosphorylation in the inner mitochondrial membrane is transferred to the microtubules to drive motility. Hence reduced sperm motility induced by cryopreservation is believed to be mainly associated with mitochondrial damage (Januskauskas, and Zillinskas, 2002; Ruiz -Pesini et al, 2001). In human spermatozoa, mitochondrial enzymatic activities were shown to be correlated with spermatozoal motility (Ruiz-Pesini et al, 2001). Male infertility can result from a significant decrease in the number of motile forms and/or from movement quality disorders. Some studies reported a significant correlation between computer assessed spermatozoal motility and field fertility (Januskauskas et al, 2003) however, in a more recent study (Garcia -Maciaset al, 2007), spermatozoal total and progressive motilities and velocity parameters were known to have no correlation with fertility though the authors noticed that velocity parameters were highest in the high-fertility group in their study. The decrease in correlation between motility and fertility has been suggested to be due to a difference in the change of permeability of the acrosome and mitochondrial membranes to calcium ions. The acrosome membrane suffers most from cold shock which accounts for most of the fertility failure (Deneke N., Lemma A., and Yilma T., 2010, Unpublished information). Vesiculation of the acrosomal and plasma membranes occurs during sperm cell death which is termed as false acrosomal reaction. A true acrosome reaction, which precedes fertilization, occurs only in live, intact spermatozoa. Thus, it is important not only to analyze semen for sperm viability but also to determine the alteration of acrosomal integrity simultaneously. The proportion of sperm cells that reacted to HOST (n=36) was 60.6±9.2% and theproportion of sperm with altered acrosome in the same sample was 50.6%. HOST reaction was highly correlated (r = 0.82, p<0.001) to higher percent live sperm in frozen sample indicating that live sperms whose membrane is relatively intact will react to the HOST solution. When the two tests are performed simultaneously, acrosomal alteration seem to be directly correlated with membrane integrity as evidenced in the correlation study). Similar correlation result between the incidence of swollen tails after incubation for min, and of altered acrosomes and total spermatozoan motility has also been previously reported for horses (Cueva et al, 1997). Pyruvate, for instance, is a potent scavenger of H2O2 and its supplementation to a chilled stored stallion semen or to a frozen thawed bull semen resulted in a significant augmentation of sperm motility and ATP levels. Oxidation of thiols in sperm proteins by O2- and H2O2 was found to be associatedwith inhibition of sperm motility and fertilizing ability (Mamoto et al, 1996). Cryopreservation of bull sperm in egg yolk based extenders significantly reduced the intracellular level of thiols and post-thaw treatment of frozen semen with thiols containing antioxidants prevented H2O2 -mediated loss of sperm motility (Bilodaeu et al, 2001). Generally, changes in plasma membrane integrity and motility are both indicators of sperm viability and metabolic intactness. In this regard boar spermatozoa are known to suffer extensive membrane and tail damage during 795

3 freezing and thawing, and those spermatozoa that survive suffer from a shortened lifespan, requiring AI to be carried out with large numbers of spermatozoa closely timed to the moment of ovulation (Wongtawan et al, 2006). Poorly motile spermatozoa are also less likely to arrive at the site of fertilization in vivo or to penetrate the zona. Moreover, the proportion of motile sperm population itself is adversely affected by cryopreservation (Cerolini et al, 2001; Gillan et al,2004). Reduced sperm binding is likely a result of membrane injury, possibly by structural damage to the sperm receptors or by incomplete receptor aggregation. Evaluation of post-thaw semen quality The semen quality and its relationship with fertility have great importance in animal production. Hence, in vitro tests are frequently applied to determine the quality of semen for its approval and use in both AI and other biotechnology procedures. Conventional laboratory tests for assessment of semen quality include light microscopic study of spermatozoal morphology, and estimation of spermatozoal motility which in turn encompass percentages of motile and progressively motile sperm; velocity of spermatozoal movement; and longevity following in vitro storage. Other features of semen quality include concentration, volume, detection of the presence of urine, blood, or potentially pathogenic bacteria and functional integrity tests. The choice of adequate parameters by reproducible, fast and sensitive methods is of increasing concern. This is because the predictive value of the standard seminal parameters is limited or insufficient for the identification of subfertile individuals (Clement, 2001; Love et al, 2000). Many tests of sperm motility, morphology, acrosomal status, defective sperm organelles and DNA, and metabolism have been correlated with fertility (Evenson et al, 2002; Larsson and Rodriquez-Martinez, 2000; Muller, 2000; Saacke et al, 2000; Thomas et al, 1998). All of these spermatozoal attributes have been shown to be either directly or indirectly affected by cryopreservation or the thawing process. The correlation between fertility and percentage of motile sperm in a semen sample has already been demonstrated. In one study, after insemination of 55 cows with frozen semen a 30.9% (17 cows) pregnancy rate with an average number of services per conception of 2.7 was found. Conception rate to first service was only 7.2%. The mean (±SD) alteration of acrosome and positive reaction to HOST for successful (pregnant) and failed insemination (non pregnant) were 47.6 ± 9.9% and 64.7 ±3.0%, and 62.7 ± 7.3% and 42.1 ± 3.9%, respectively with a highly significant (p<0.001) difference in both tests between the successful and failed inseminations. Semen that did not impregnate contained the highest proportion of sperm with altered acrosome that did not react well to HOST. This shows the significance of the use of combination of semen evaluation methods to avoid the use of poor quality semen and hence reduce the male factor from fertility assessments (Deneke N., Lemma A., and Yilma T., 2010, Unpublished information). Significant variation in the success rates for frozen semen has been reported with an apparently much wider variation in the performance of equine semen post freezing and thawing than is with bovine semen. For instance, one-cycle pregnancy rates of 32-51% have been reported in mares (Muller, 1987; Pickett and Amann, 1993). The rate of temperature drop was found to be most critical over the specific temperature range of 0-5 C when motility was evaluated later. In general, the faster the rate of cooling, the more severe is the damage (Kayser, 1990). There is further evidence which suggests that the rate of temperature drop also determinesthe subsequent active life of the spermatozoa (Andrabi, 2007). DISCUSSION The lack of significant differences between treatments, in this study, on sperm motility agrees with Vadnais et al. (2005) results, who also did not find any effect on this variable using different SP treatments during the thawed boar semen process. Likewise, Okasaki et al. (2009) did not find effect of the addition of 10% and 20% SP on boar semen motility after 6 hours of incubation. On other hand, Satorre et al. (2007) found a significant improvement in sperm motility with the addition of VE to the freezing medium. Peña et al. (2003) evaluated two levels of VE in two fractions of the ejaculate and observed that the motility observed significantly improved in comparison with the control group. Hernández et al. (2007b) reported a significant improvement in sperm motility with the addition of SP to the boar semen freezing medium. Garcia et al. (2010) found a significant effect of treatment (P<0.001) on motility, when post-thawed porcine semen was incubated with 10% SP at 0 and 60 min (48 and 28%, respectively). Similar results using VE were reported by Breininger et al. (2005). It is important to mention that the subjective evaluation process of motility is not a reliable indicator of the quality of the sperm after thawing. There are discrepancies between the results reported by various authors and the obtained in this study. One of the possible explanations is the reliability of the results, characterized by sensitivity and the specificity of method of evaluation (optic microscope). When this subjective method is used, it is estimated 30-60% variation compared with objectives methods, such as Computer Assisted Sperm Analysis, CASA (Budworth et al., 1988; Amman, 1989; Peña et al., 2003). Results of this study indicate ph was not a reliable measurement in predicting the protective qualities of the extender for the sperm during cryopreservation. The 20% and 30% egg yolk concentration extenders were optimal in comparison to the remaining extenders in their final postthaw motility and morphology though their initial ph represented the highest and lowest measurements, 7.12±0.04 and 7.022±0.04 respectively.casa has high levels of accuracy and reliability when compared with other methods of sperm classification (Verstengen et al., 2002). percentage of intact acrosomes when they added SP during the freezing boar semen process. The results of this study suggest that the dose of Vitamin E (α - tocopherol) was enough to inhibit the effects of lipid peroxidation by enhancing the acrosomal integrity in the thawed semen. This could be explained because α-tocopherol protects the spermatozoa of detrimental effects caused by the oxidative stress associated to the thawing process. It has been expressed that VE inhibit the lipid peroxidation by removing the free radicals before they act on the fatty acids of the sperm plasma membrane contributing to the maintenance of sperm viability (Merkies et al., 2003; Breininger et al., 2005; Ordoñez, 2008). Moreover, we observed a significant decrease in the post-thaw percentage of viable spermatozoa with intact acrosomes in both control and treated specimens. In normal semen, a induced protective effect on acrosome loss during the freeze-thaw process was observed (12). In the present study, we evaluated poor quality semen, and it is possible that 796

4 normal semen would resist cryoinjury better than semen obtained from oligoasthenozoospermic men. We observed that prefreezetreatmentof sperm from such individuals with PF improved the ability of cryo-thawed spermatozoa to undergo the acrosome reaction in response to stimuli. Interestingly, the ability of cryo-thawed sperm from abnormal specimens to respond to ionophore challenge appears to be lower than that of normal sperm, irrespectively of treatment with PF before freezing (12). During post-cool evaluation of the various extenders no differences were found in the motility between extenders. This observation was anticipated, as there are canine semen extenders commercially available void of egg yolk. An unexpected result was the significant 11% decrease of normal morphology in the 0% egg yolk concentration extender after 3 hours of cooling at 5 C with no significant change in the remaining extenders. Table1.SampleSpecifications Average mobility Mobility SD The lowest mobility Maximum Mobility Inter-quartile range Average CSF after thawing ± ± ± ± ± ±6.26 (n = 22) Average mobility in samples with motility> 50% (n = 5) Average CSF mobilization ± ± ± ± ± ±5.32 in samples with> 50% (n = 5) Average mobility in samples with motility <50% (n = 5) Average CSF mobilization in samples with <50% (n = 5) ± ± ± ± ± ±6.11 Table2. General freezing program Step From ( o C) To ( o C) Rate ( o C/min) Duration (s) Table3. Final Software Results Medium Type % Live % Acrosome % Motile VAP (m/s) VSL (m/s) VCL (m/s) 1 T T T T T T T T S S S S S S S S Figure1.Software Results 797

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