پديد آورندگان :
نظري، مهدي دانشگاه تبريز - دانشكده كشاورزي - گره علوم دامي , دقيق كيا، حسين دانشگاه تبريز - دانشكده كشاورزي - گره علوم دامي , امامي، جميله دانشگاه تبريز - دانشكده كشاورزي - گره علوم دامي
كليدواژه :
آنتي اكسيدان , اسپرم , سيستئامين , تنش اكسيداتيو , مالون دي آلدهيد , خروس
چكيده فارسي :
آسيب غشايي بعنوان يكي از دلايل كاهش تحرك و باروري اسپرم خروس مطرح است. تاكنون آنتي‌اكسيدان‌هاي مختلفي جهت كاهش يا جلوگيري از آسيب‌هاي غشايي در حين انجماد و ذوب مورداستفاده قرارگرفته است. هدف: در اين تحقيق از اسيدآمينه سيستئامين بمنظور كاهش استرس اكسيداتيو، پراكسيداسيون ليپيدي و بهبود عملكرد اسپرم طيور استفاده شد. روش كار: بدين منظور نمونه‌هاي مني از 15 خروس نژاد رأس با سن 28 هفته به روش مالش پشتي-شكمي جمع‌آوري شدند. نمونه‌هاي مني پس از ارزيابي اوليه باهم مخلوط شده پس از رقيق‌سازي نمونه‌ها و افزودن سطوح مختلفي از اسيدآمينه سيستئامين، نمونه‌ها سردسازي شده و سپس فريز شدند. بعد از ذوب نمونه‌ها فراسنجه‌هاي حركتي اسپرم با استفاده از سيستم آناليز كامپيوتري اسپرمCASA، زنده‌ماني با استفاده از روش رنگ‌آميزي ائوزين-نيگروزين، يكپارچگي غشاء با استفاده از آزمون هاست، ميزان اسپرم‌هاي ناهنجار با استفاده از محيط هانكوك و ميزان ليپيد پراكسيداسيون اندازه‌گيري شدند. نتايج بر اساس نتايج بدست‌آمده افزودن سطوح 30 و 45 ميكرومول باعث افزايش تحرك كل نسبت به گروه كنترل گرديد. افزودن سطح 45 ميكرومول باعث افزايش تحرك پيش‌رونده و پارامترهاي حركتي و همچنين افزودن 30 ميكرومول باعث افزايش معني‌دار ميزان زنده‌ماني، سلامت غشاء و كاهش معني‌دار ميزان مالون‌دي‌آلدهيد نسبت به گروه كنترل شد. افزودن اسيدآمينه سيستئامين باعث كاهش معني‌دار اسپرم‌هاي با مورفولوژي ناسالم طي انجماد-يخ‌گشايي نشد.
افزايش تحرك پيش‌رونده و پارامترهاي حركتي و همچنين افزودن 30 ميكرومول باعث افزايش معني‌دار ميزان زنده‌ماني، سلامت غشاء و كاهش معني‌دار ميزان مالون‌دي‌آلدهيد نسبت به گروه كنترل شد. افزودن اسيدآمينه سيستئامين باعث كاهش معني‌دار اسپرم‌هاي با مورفولوژي ناسالم طي انجماد-يخ‌گشايي نشده است. نتيجه گيري نهايي: مطالعه حاضر نشان داد افزودن
سيستئامين ميتواند باعث محافظت اسپرم از تنش اكسيداتيو گردد و پراكسيداسيون ليپيد را كاهش بدهد.
چكيده لاتين :
Introduction: Long-term storage of semen is essential for achieving the benefits of artificial insemination (Tuncer et al. 2010). This is carried out by sperm cryopreservation, which stops the sperm metabolic activities, allowing long storage (Bailey et al. 2000). This process affects the sperm quality (Wang et al. 1991) by introducing mechanical and chemical damage, production of reactive oxygen species, oxidative stress, and reducing antioxidant activity (cysteamine is an aminothiol antioxidant as an effective scavenger). Cysteamine is known to have been reported in some studies to improve the freezing of ram sperm (Bucak et al. 2007). The aim of the present study was to determine the antioxidant effects of cysteamine on the functional parameters of cysteamine in Lake Extender based on soybean lecithin.
Material and methods This study was carried out in University of Tabriz research station. For this purpose, 15 adult roosters (25 weeks old) were used. Sperm collection was done by dorsal-abdominal massage. The roosters were habituated for one month and sperm collection was performed twice a week. First, sperm were examined for volume, concentration and color, and only samples with volume of 0.2 to 0.7 mL and motility ≥80% and concentration above 3×109 were used. To eliminate the individual effects, the confirmed samples were pooled. Four levels containing control, 0.15, 0.30, and 0.45 μM cysteamine were then added to Lake Extender containing (one part of semen and four of extender). The cooling process was carried out in two steps. The samples were adjusted to 4°C for two hours. Samples were transferred to the refrigerator for one more hour, then they were drawn into 0.25 mL straws, placed 4 cm above nitrogen vapor for 7 min, and immersed in liquid nitrogen. They were stored in liquid nitrogen until further analysis. For assessment, the cryopreserved straws were thawed in a water bath at 37 °C for 30 s. The motility parameters were evaluated using CASA, viability by Eosin-Nigrosin staining, membrane integrity by Host tests, sperm abnormality by Hancock test and lipid peroxidation by MDA.
Results and discussion: Based on the results (Table 2), addition of 30 and 0.45 μM significantly increased total motility and 0.30 μM level improved progressive motility, VAP, VSL and VCL parameters (P <0.05). The results show that the addition of cysteamine amino acid improves sperm quality. Addition of 0.30 μM level significantly increased viability and plasma membrane integrity of rooster sperm (P <0.05) while significantly decreasing malondialdehyde compared with the control group (P <0.05). The addition of cysteamine insignificantly reduced sperm abnormality. The aim of this study was to reduce the production of ROS and the rate of lipid peroxidation while improving membrane health and survival. Bird sperm has an advanced antioxidant system that includes the antioxidants glutathione, peroxidase, and superoxide dismutase (Surai 1999). Adding antioxidants can control ROS production (Amini et al. 2015). Antioxidants in different species and at different doses have shown different results (Nazari and Daghigh kia 2020). Addition of cysteamine to rooster spermatozoa during freezing and thawing significantly increased motility and survival parameters, which agreed with the results of Bucak et al. (2007). Najafi et al. (2014) indicated that cysteamine at 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). cysteamine improved membrane functionality significantly, except at 8 mM.n. This was probably due to the high dose of diluent used. Our results did not agree with the study of Thananurak et al. (2019) who reported the negative effect of adding levels of 0.001, 0.002 and 0.004 μM on motility and viability. Low levels of cysteamine induce cysteine to enter the cell, producing glutathione as an intracellular antioxidant. Cysteamine at high doses produces large amounts of hydrogen peroxide, causing oxidative stress and reducing glutathione peroxidase activity (Besouw et al. 2013). According to report Partyka et al. (2013) cysteine increases motility and viability of rooster sperm. Glutathione peroxidase is one of the enzymes with peroxidative activity that plays an important role in sweeping high oxidations and protecting the cell from oxidative stress. Glutathione can also restore the oxidized vitamins E and C and restore them to the original antioxidant structure (Almasi et al. 2014). Cysteamine as a glutathione synthase can play a major role in reducing free radicals. In the present study, the amino acid cysteamine decreased malondialdehyde concentration and increased plasma membrane integrity and normal sperm during the freezing-thawing process. This agreed with the result of Najafi et al. (2014) reporting that addition of 6 mM reduced the concentration of malondialdehyde in ram semen compared with the control group. Not only does cysteamine improve sperm quality after freeze-thawing, but also it increases sperm resistance during artificial insemination in the reproductive female tract (Najafi et al. 2014). In the present study, cysteamine did not significantly decrease the percentage of abnormal sperm compared with the control group.
Conclusion According to the results of this study, adding amino acid cysteamine to the diluted sample of semen can protect sperm from oxidative stress and reduce lipid peroxidation. Levels of 0.30 and 0.45 μM increase the mobility. Also, the level of 0.30 μM increases the viability and health of the sperm membrane and reduces the amount of malondialdehyde in the rooster's semen.
