The effect of cysteine on post-thawed buffalo bull (Bubalus Bubalis) sperm parameters.
Buffalo population of the world was estimated to be more than 140 million . Artificial insemination is one of the important procedures, which causes the widespread propagation of semen, limiting the spread of sexually transmitted diseases, and chiefly facilitating genetic improvement programmes  Nevertheless, cryopreservation generates sublethal injury to the sperm due to chemical, osmotic, thermal, and mechanical stresses, which may result in loss of viability, motility, damage of desoxyribonucleic acid (DNA), destruction of acrosomal and plasma membrane . Furthermore change of biochemical factors have been recognized during cryopreservation, including depletion of amino acids and lipoproteins, release of glutamic-oxaloacetic transaminase (GOT), decrease in phosphatase activity, decrease in loosely bound cholesterol protein, inactivation of acrosin enzyme and hyaluronidase, prostaglandins diminution, increase in sodium, decrease in potassium content, reduction of ATP and ADP synthesis and decrease in acrosomal proteolytic activity . The extenders is an important factor in cryopreservation process. These mediums must have adequate pH and buffering capacity, appropriate osmolality and should protect spermatozoa from cryogenic lesion. The Tris extenders are an important mediums that often used for semen freezing of bulls, rams, bucks and buffaloes [2,11].
In fact sperm plasma membrane is a primary target for freezing or cold shock injury . Amino acids have important roles in preventing cryogenic damage in bovine, buffalo, goat and Ram's spermatozoa during the frozen stage [10,13].
The cells of Mammalian can use cysteine that it enhanced intracellular glutathione synthesis . Cysteine has been demonstrated to contract with reduction of motility, viability and membrane integrity during sperm storage .
Material and methods
The anti-oxidants used in this study such as Cystein, and other chemicals were obtained from Sigma-Aldrich.
2.2. Animals, Semen Collecting and Processing:
The four mature buffalo-bulls (3-4 years of age, and 600-650 kg of body weight) maintained at the northwest buffalo Research Institute, Ministry of Agriculture, Urmia, Iran, under uniform feeding, housing and lighting conditions were used as a semen donor. A total number of 20 ejaculates were collected from buffalo-bulls with the aid of an artificial vagina at weekly intervals for 5 weeks. Immediately after collection, the ejaculates were transferred to the laboratory, and evaluated for sperm motility and sperm concentration. The sperm concentration of each ejaculate was determined by means of a spectrophotometer. Only ejaculates containing sperm with >80% progressive motility and a concentration higher than 2.5 x [10.sup.9] sperm/ml were pooled, in order to eliminate the bull effect. The base extender was Tris-based extender (Tris 2.66 g/100 ml, citric acid 1.47 g/100 ml, fructose 0.63 g/100 ml, egg yolk 20% (v/v), glycerol 7% (v/v), pH 6.8) was used as the basis extender. Each mixed ejaculate was split into 11 equal aliquots and diluted at 37[degrees]C to concentration of 6 x [10.sup.6] sperm/mL with the base extender containing cysteine (0.1, 1,2.5, 5 or 7.5 mM), Vit E (0.1, 0.5, 1 or 1.5), and no anti-oxidants (control), respectively. Diluted semen samples were aspirated into 0.5 ml French straws, sealed with polyvinyl alcohol powder and cooled horizontally from 37 to 4[degrees]C, and maintained at 4[degrees]C for 4 hrs. The straws were frozen in vapor 4cm above liquid nitrogen for 10 minutes and plunged into liquid nitrogen. After being stored for one month, 5 frozen straws from each group were thawed individually at 37[degrees]C for 20sina water bath for microscopic evaluation.
2.3. Semen Evaluation:
2.3.1. Evaluation of Sperm Parameters:
* Sperm Viability:
A 50-[micro]L drop of frozen-thawed semen was placed on a pre-warmed slide and mixed with 50 [micro]l drop of the supravital stain [1% (w/v) eosin B, 5% (w/v) nigrosin in 3% tri-sodium citrate dehydrate solution] to prepare a thin smear . After air-drying, the smear was evaluated by microscope with 400x magnification. Two hundred spermatozoa were counted for unstained heads of spermatozoa (live) and/or stained/partial stained heads of spermatozoa (dead) .
* Post-thawed Sperm Motility:
Percentage motility was assessed using a phase-contrast microscope (x40). A 5-[micro]L drop of thawed semen placed directly on a pre-warmed slide and covered with a cover slip. Sperm motility estimations were performed in three different microscopic fields for each semen sample by the same researcher. The mean of three observations was considered as a single data point.
* Normal Acrosomes:
A 500-[micro]L portion of each semen sample was fixed in 50 [micro]L of a 1% solution of formal citrate containing 2.9% (w/v) trisodium citrate dehydrate (Merck) and 1% (v/v) commercial formaldehyde. Two hundred spermatozoa were counted with a phase contrast microscope (1000x) for their normal apical ridge.
The study was replicated three times. Results are expressed as the mean [+ or -] standard error of the mean (SEM). Analysis of variance (ANOVA) was used to assess differences among treatments on motion characteristics, plasma membrane integrity and normal acrosome morphology. When the F ratio was significant (P<.05), Tukey's post hoc test was used to compare treatment means (Version 12.0; SPSS, Chicago, IL).
* Sperm Parameters (Percentages of Motility, Viability, Acrosome Normality):
The effects of anti-oxidants on the sperm characteristics of frozen buffalo semen were evaluated in the six independent experiments.
The post-thaw sperm motility improved significantly () in the group as compared to their respective values
As set out in Table 1, the the inclusion of cysteine led to a higher motility, compared to the control group (P < 0.05).
The motility of sperm cells post thaw increased significantly when adding of 2.5, 5 and 7.5 mM Cysteine (57.90 [+ or -] 3.13, 58.20 [+ or -] 0.14 and 59.10 [+ or -]0.16 respectively) to the extender as compared to the control and 0.1, 1 mM Cysteine groups (45.86[+ or -] 0.36, 44.91 [+ or -] 0.36 and 52.95[+ or -] 0.15 respectively). No significant differences were observed in acrosome damage, following the supplementation of the freezing extender with anti-oxidants, following the freeze-thawing process.
The viability of sperm cells post thaw increased significantly when adding of 1, 2.5, 5 and 7.5 mM Cysteine (59.43 [+ or -] 0.19, 57.90 [+ or -] 3.13, 58.20 [+ or -] 0.14 and 59.10 [+ or -] 0.16 respectively) to the extender as compared to the control group (P<0.05) and the highest viability of sperms were seen at 7.5 mM Cysteine treatment. Also, lower level of cysteine cause declining of live sperm percent, that it is not significant compred to to the control group.
Improvement of semen freezing methods require to having knowledge about occurred biochemical and physiological processing during the freezing, thawing and extender characteristics [4,612]. Antioxidant capability in sperm cells is limited because of deficiency cytoplasmic components having antioxidant effects to expunction of reactive oxygens. Thus, mammal's sperm haven't enough ability to encountering with peroxidation during the freezing and thawing processes [1,3,7]. Reactive oxygen are responsible to sperm membrane damage and directly damage sperm DNA that cause reduces of the sperm's motility, acrosomal membrane integrity and sperm metabolic alterations. In recent years, adding antioxidants to semen extenders for improvement of sperm quality were studied.
Addition of additives such as cysteine and lipoic acid to the semen freezing extender, may prevent cryodamage to spermatozoa metabolism and antioxidant capacities.
In recent years cysteine is one of the additives that have been used in freezing extender of human, boar, goat, bull to improve past-thaw sperm parameters.4
In the present study, the inclusion of different levels of cysteine to extender before freezing causes significant improvement in sperm quality parameters such as motility and viability of sperms than control group (P<0.05). These findings were similar to results obtained with Sariozkan et al. . Current findings are in contrary with those reported by Michael et al.  who demonstrated L-cysteine had very limited positive effects on chilled canine semen parameters.
However, we haven't seen significant differences in normal acrosom percent between treatment and control groups. Only in treatment of 5 mM of cysteine were seen increases in normal acrosomal percent, that it was not significant. This finding was in contrast with results of Sariozkan et al.  that may be due to the differences in semen extender and animal species.
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(1) Rahim Beheshti., (3) Aiden Asadi., (1) Behrad Eshratkhah., (2) Jamshid Ghiasi GhaleKandi., (2) Abolfazl Ghorbani
(1) Department of veterinary, Shabestar branch, Islamic Azad University, Shabestar, Iran.
(2) Department of Animal Science, Shabestar branch, Islamic Azad University, Shabestar, Iran.
(3) Department of Agriculture, Shabestar branch, Islamic Azad University, Shabestar, Iran.
RahimBeheshti., Department of veterinary, Shabestar branch, Islamic Azad University, Shabestar, Iran.
Table 1: Effect of of different concentarations of cysteine on buffalo-bull semen Motility after freezing-thawing. Treatments Motility (%) Cysteine 0.1 mM 44.91 [+ or -] 0.36 (c) Cysteine 1 mM 52.95 [+ or -] 0.15 (b) Cysteine 2.5 mM 57.90 [+ or -] 0.13 (a) Cysteine 5 mM 58.20 [+ or -] 0.14 (a) Cysteine 7.5 mM 59.10 [+ or -] 0.16 (a) Control 45.86 [+ or -] 0.36 (c) Values are (mean [+ or -] standard error of mean). Different letters within a column indicates significant differences (P<0.05). Table 2: Effect of of different concentarations of cysteine on buffalo-bull semen acrosomal normality after freezing-thawing. Treatments Acrosomal normality (%) Cysteine 0.1 mM 67.85 [+ or -] 0.52 Cysteine 1 mM 68.10 [+ or -] 0.76 Cysteine 2.5 mM 67.98[+ or -] 0.11 Cysteine 5 mM 68.46[+ or -] 0.20 Cysteine 7.5 mM 67.85[+ or -] 0.52 Control 68.38[+ or -] 0.23 Values are (mean [+ or -] standard error of mean). Table 3: Effect of of different concentarations of cysteine on buffalo-bull semen viability after freezing-thawing. Treatments Live sperm (%) Cysteine 0.1 mM 55.71 [+ or -] 0.20 e Cysteine 1 mM 59.43 [+ or -] 0.19 d Cysteine 2.5 mM 66.15 [+ or -] 0.24 c Cysteine 5 mM 68.26 [+ or -] 0.20 b Cysteine 7.5 mM 69.20 [+ or -] 0.24 a Control 51.55[+ or -] 0.33 e Values are (mean [+ or -] standard error of mean). Different letters within a column indicates significant differences (P<0.05).