Effect of Antioxidant D-Aspartic Acid and Thawing Rate on the Freeze-Thawing Process of Ram Semen
محورهای موضوعی : Camelح. دقیق کیا 1 , ص. وطن‌خواه 2
1 - Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
2 - Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran
کلید واژه: ram, cryopreservation, antioxidants, D-aspartic acid, thawing rate,
چکیده مقاله :
The study was conducted to determine the influence of D-aspartic acid (D-Asp) as antioxidant supplement and thawing rates on ram sperm motility, membrane integrity, abnormality, viability, mitochondria activity, malondialdehyde and antioxidant activities, and total antioxidant capacity after freezing-thawing process. Semen samples from five mature rams (3-4 years old) were diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and D-Asp (5, 10, and 15 mg/L) and cryopreserved. Frozen straws were thawed at water bath temperatures at 37 ˚C for 30s and at 60 ˚C for 6s. Addition of 10 mg/L of D-Asp improved significantly progressive motility, average path velocity, and straight-line velocity percentages after freeze-thaw (P<0.05). Plasma membrane integrity, mitochondria activity, viability, total antioxidant capacity, and glutathione peroxidase were higher in group receiving 10 mg/L in comparison to other treatments (P<0.05). Meanwhile, total abnormality significantly decreased at this concentration (10 mg/L) in comparison to the level of 15 mg/L and control group (P<0.05). Our results revealed that malondialdehyde level was lower in group receiving 10 mg/L D-Asp compared to other treatments (P<0.05). There were no significant interactions between concentrations of D-Asp and thawing protocols for any semen samples. Also, significant improvement in sperm viability and mitochondria activity were observed at 37 ˚C to 30 s thawing method (P<0.05). In overall, results of the present study demonstrate that addition of D-Asp at level of 10 mg/L have beneficial effect on quality of post-thawed ram semen cryopreserved in an extender. Therefore, this antioxidant in the suitable dose can be recommended as an additional component of ram freezing extender. The 60 ˚C to 6s thawing procedure is not an appropriate replacement for 37 ˚C to 30 s.
این مطالعه به منظور تعیین اثر D-آسپارتیک اسید (D-Asp) به عنوان مکمل آنتیاکسیدانی و سرعت یخگشایی بر تحرک، یکپارچگی غشاء، ناهنجاری، زندهمانی، فعالیت میتوکندریایی، میزان مالون دی آلدهید، فعالیت آنتیاکسیدانی و ظرفیت کل آنتیاکسیدانی بعد از فرآیند انجماد–یخگشایی اسپرم قوچ انجام گرفت. نمونههای منی از پنج رأس قوچ بالغ (4-3 ساله) گرفته شده و با استفاده از رقیقکننده (5/1 درصد لستین سویا و 7 درصد گلیسرول)، بدون آنتی اکسیدان (شاهد) و دارای D-Asp (5، 10 و 15 میلیگرم در لیتر) محافظت انجمادی شدند. پایوتهای منجمد شده با استفاده از حمام آب گرم با دمای °C 37 در مدت 30 ثانیه و در دمای °C 60 به مدت 6 ثانیه یخگشایی شدند. نتایج نشان داد که افزودن 10 میلیگرم بر لیتر از D-Asp باعث بهبود معنیدار درصد حرکت پیشرونده، میانگین مسیر طی شده (VAP) و سرعت در خط مستقیم (VSL) اسپرمها بعد از فرآیند انجماد-یخگشایی شد (05/0>P). میزان یکپارچگی غشاء پلاسمایی، فعالیت میتوکندریایی، زندهمانی، ظرفیت کل آنتیاکسیدانی و فعالیت گلوتاتیون پراکسیداز در نمونههای دریافت کننده10 میلیگرم در لیتر D-Asp نسبت به دیگر تیمارها بالاتر بود (05/0>P). در همین حال، ناهنجاری کل در نمونههای دریافت کننده 10 میلیگرم در لیتر D-Asp نسبت به 15 میلیگرم در لیتر و یا گروه کنترل کاهش معنیداری داشت (05/0>P). نتایج این آزمایش نشان داد که سطح مالون دی آلدهید در گروه دریافت کننده 10 میلیگرم در لیتر D-Asp نسبت به دیگر تیمارها کمتر بود (05/0>P). اثر متقابل معنیداری بین غلظتهای مختلف D-Asp و متدهای مختلف ذوب منی منجمد مشاهده نشد. استفاده از روش ذوب منی در دمای °C 37 و در مدت 30 ثانیه باعث بهبود معنیدار زندهمانی اسپرم و فعالیت میتوکندریایی شد (05/0>P). به طورکلی، نتایج مطالعه حاضر نشان داد که افزودن 10 میلیگرم در لیتر D-Asp اثرات مفیدی بر روی کیفیت منی قوچ بعد از انجماد و یخگشایی میشود. بنابراین افزودن این آنتی اکسیدان در مقادیر مناسب به عنوان یکی از اجزای رقیقکنندههای انجمادی منی قوچ قابل توصیه میباشد. استفاده از پروتکل دمای ذوب °C 60 به مدت 6 ثانیه نمیتواند جایگزین مطلوبی با پروتکل ذوب منی منجمد در دمای °C 37 و در مدت 30 ثانیه باشد.
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