سابقه و هدف: در تلقیح مصنوعی حیوانات مزرعه‌ای معمولا از اسپرم منجمد استفاده می‌شود. یکی از روش‌های مهم جهت حفظ و نگهداری ذخایر ژنتیکی انجماد اسپرم است که به عنوان یکی از روش‌های کمک باروری می‌تواند سبب حفظ کیفیت اسپرم شود. از ابتدای بکارگیری تکنیک تلقیح مصنوعی، رقیقکنندههای مختلفی جهت حفظ و نگهداری منی در شرایط مایع و منجمد استفاده شده است. ترکیب و ماهیت رقیق‌کننده‌ها از اهمیت زیادی در زنده‌مانی اسپرم برخوردارند. بدین منظور هدف از این پژوهش بررسی اثرات رقیقکنندههای شیر، تریس-زرده تخم‌مرغ و تریس-زرده تخم بوقلمون و سطوح مختلف قند تره‌هالوز (صفر، 80، 100 و 120 میلی‌مولار) بر کیفیت منی قوچ دالاق پس از فرآیند انجماد-یخ‌گشایی بود. مواد و روش‌ها: در این پژوهش نمونه‌های منی از چهار رأس قوچ دالاق 2 تا 3 ساله با استفاده از مهبل مصنوعی جمع‌آوری گردید. سپس نمونه‌ها در رقیقکنندههای مختلف، رقیق‌سازی شدند و تیمارهای مختلف قند تره‌هالوز را دریافت نمودند. نمونه‌های منی وارد پایوت‌های 5/0 میلی‌‌لیتری شدند و به مدت 150 دقیقه در دمای 4 درجه سانتی‌گراد خنک شدند و سپس در نیتروژون مایع منجمد گردیدند. پایوت‌های منجمد شده برای ذخیره‌سازی به تانک حاوی ازت منتقل شدند. پس از یخ‌گشایی فراسنجه‌های کیفی اسپرماتوزوآ ﺷﺎﻣﻞ درﺻﺪ تحرک اسپرم، زنده‌مانی، سلامت غشاء و اسپرم طبیعی ﻣﻮرد ارزﯾﺎﺑﯽ ﻗﺮار ﮔﺮﻓﺘﻨﺪ. این آزمایش به صورت فاکتوریل 3×4 با استفاده از طرح کاملا تصادفی انجام شد. تیمارها شامل سطوح مختلف قند تره‌هالوز (صفر، 80، 100 و 120 میلی‌مولار) و رقیقکنندههای مختلف (شیر، تریس-زرده تخم‌مرغ و تریس-زرده تخم بوقلمون) بودند. داده‌های به دست آمده از این پژوهش با استفاده از نرم افزار آماری SAS (مدل 1/9) با رویه ANOVA مورد تجزیه و تحلیل قرار گرفتند. مقایسه میانگین داده‌ها توسط آزمون توکی در سطح خطای 1 درصد انجام شد. یافته‌ها: نتایج پژوهش حاضر نشان داد که تأثیر سطوح مختلف قند تره‌هالوز بر درصد تحرک اسپرم، زنده‌مانی، سلامت غشاء و اسپرم طبیعی معنی‌دار بود (01/0P<). بیشترین تحرک اسپرم (59/1±33/42 درصد)، زنده‌مانی (61/1±73/68 درصد)، سلامت غشاء (35/1±06/66 درصد) و اسپرم طبیعی (25/1±66/79 درصد) در سطح 120 میلی‌مولار قند تره‌هالوز بدست آمد. تأثیر رقیق‌کننده‌ها نیز بر درصد تحرک اسپرم، زنده‌مانی، سلامت غشاء و اسپرم طبیعی معنی‌دار بود (01/0P<). بیشترین تحرک اسپرم (38/1±50/42 درصد)، زنده‌مانی (39/1±25/71 درصد)، سلامت غشاء (17/1±30/53 درصد) و اسپرم طبیعی (08/1±85/73 درصد) در رقیق‌کننده تریس-زرده تخم بوقلمون بدست آمد. نتیجه‌گیری: در کل، با در نظر گرفتن همه شاخص‌های ارزیابی شده در رقیق‌سازی منی قوچ دالاق، افزودن 120 میلی‌مولار قند ترهالوز در رقیق‌کننده تریس-زرده تخم بوقلمون پاسخ بهتری نسبت به دیگر تیمارها پس از فرآیند انجماد-یخ‌گشایی نشان داد.
The effect of different extenders and different levels of trehalose sugar on quality of Dalagh ram semen in frozen condition
چکیده [English]
Background and objectives: In artificial insemination of farm animals, frozen sperm is usually used. Sperm cryopreservation is one of the important methods for maintain and preserve genetic resources, which as one of the methods of assisted reproductive, can maintain the quality of sperm. Since beginning the time of artificial insemination technique, different diluents have been used to maintain and preserve semen in liquid and frozen condition. Extender composition and nature are of great importance in the viability of sperm. To this end, the aim of this research was to evaluate effects of milk, Tris-chicken egg yolk and Tris-turkey egg yolk extenders and different levels of trehalose sugar (0, 80, 100 and 120 mM) on Dalagh ram semen quality after freezing-thawing process. Materials and methods: In this research semen samples were collected from four Dalagh rams in the age of 2-3 years using artificial vagina. Then samples were diluted in different extenders and received different trehalose sugar treatments. Semen samples were aspirated into 0.5 ml straws and cooled to 4°C within 150 min and then frozen in liquid nitrogen. Frozen straws were transferred to a tank containing liquid nitrogen. After thawing spermatozoa quality parameters including percentage of sperm motility, viability, membrane integrity and sperm normality were evaluated. This experiment was carried out in 4×3 factorial arrangement with the use of completely randomized design. Treatments were included different levels of trehalose sugar (0 , 80 , 100 , 120 mM) and different extenders (milk and Tris-chicken egg yolk and Tris-turkey egg yolk). The obtained data from this study were analyzed by ANOVA procedure of Statistical Analysis System SAS (version 9.1). Comparison of data means were performed by Tukey test at the level of %1 error. Results: The results of this research showed effects different levels of trehalose sugar, on the percentage of sperm motility, viability, membrane integrity and sperm normality were significant (P<0.01). The highest sperm motility (42.33±1.59%), viability (68.73±1.61%), membrane integrity (66.06±1.35%) and sperm normality (79.66±1.25%) were obtained in 120 mM trehalose sugar. The effects extenders, on the percentage of sperm motility, viability, membrane integrity and sperm normality was significant (P<0.01). The highest sperm motility (42.50±1.38%), viability (71.25±1.39%), membrane integrity (53.30±1.17%) and sperm normality (73.85±1.08%) were obtained in Tris-turkey egg yolk extender. Conclusion: In general, considering all the indices evaluated for the dilution of Dalagh ram semen, the addition of 120 mM trehalose sugar in Tris-turkey egg yolk extender showed a better response than other treatments after freezing-thawing process.
کلیدواژه‌ها [English]
Frozen, Trehalose, Extender, Semen, Ram Dalagh
مراجع
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