كليدواژه :
اسپرم خروس , انجماد , يخ گشايي , آنتي اكسيدان كوركومين , راديكال هاي آزاد
چكيده لاتين :
Introduction:The most worthwhile technique for preservation and management of bird genetic resources is semen cryopreservation, which has been studied in domestic birds like chicken. Despite years of intensive investigations, still more work should be done in order to perform successful cryopreservation of poultry sperm. The lower quality of frozen-thawed poultry sperm and consequently the poor fertilization rates compared to mammalian species are due to the exceptional morphological properties of poultry sperm, which cause freeze damages because of their vulnerable structure to low temperatures. During sperm cryopreservation, it is exposed to cold and osmotic shock, and resulted in increasing oxidation due to raise in oxidative reactions. It reduces sperm motility, viability, and ultimately reduces fertility. Oxidative stress occurs when oxidants are more potent than antioxidants. Hence, increased production of reactive oxygen species (ROS) caused by oxidative stress leads to lipid peroxidation (LPO), apoptosis, and DNA damage. Generally, the most important effect of lipid peroxidation on cells is the disruption of membrane structure and function. Plasma membrane damage is considered as one of the reasons for decreasing motility and fertility of rooster sperm. The aim of this study was to investigate the effects of different levels of curcumin antioxidants (0, 100, 200 and 300 μM) in Beltsville modified diluents for the cryopreservation of rooster semen. The present hypothesis was that curcumin antioxidants would be effective during cryopreserving of rooster sperm. Several parameters such as sperm motility, abnormalities, membrane integrity, viability and abnormality were assessed in this study to find the best level of curcumin antioxidants for cryopreservation of rooster sperm.
Material and methods: Semen samples were collected from eight Ross 308 rooster three times a week by massaging along the backbone and abdomen .The criteria in normal quality of sperm was as follows: the volume between 0.2 and 0.6 ml (semen volume was measured visually using a graduated collection tube); the concentration of sperm ≥ 3˟109 sperm/ml (ejaculate concentration was evaluated by haemocytometer); total motility ≥80% and abnormal morphology (Hancock method [17]) ≤ 10%. Then, the semen samples were pooled to remove individual variations and obtain sufficient sperm for analysis. Different levels of curcumin were added to semen samples and followed by freezing. After thawing following sperm parameters were evaluated: total motility (TM, %), progressive motility (PM, %), average path velocity (VAP, μm/s), curvilinear velocity (VCL, μm/s), straight linear velocity (VSL, μm/s), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz), straightness (STR, %), and linearity (LIN, %) using CASA software, viability by Eosin-Nigrosine Staining, membrane integrity by HOST test, and sperm abnormality by Hancock test. .
Results and discussion: The freezing extender supplemented with 200 μM of curcumin resulted in higher percentages of total and progressive motilities in comparison with other groups and control group following the freeze-thawing process (P <0.05). Group receiving 200 μM of curcumin had better performances in terms of VAP, VSL, VCL, and LIN. The analysis demonstrated that the samples supplemented with 200 μM of curcumin could improve sperm viability percentage (P< 0.05). It can be concluded that curcumin, having antioxidant properties, protects sperm from oxidative stress damage and has a positive effect on sperm viability. Samples with 300 μM curcumin had the highest amount of plasma membrane integrity and lowest amount of abnormality compared to other treatments and the control group (P< 0.05). Since the use of curcumin prevents the formation of ROSs during freezing, it therefore improves the normal morphology of sperm after thawing. One of the possible mechanisms in which curcumin improves motility and survival parameters is its antioxidant properties in preventing the formation of free radicals or scavenging it and reducing oxidative stress. No significant effect was noted on the STR parameter between different groups.
Conclusion: The results of this study showed that adding 200 μM curcumin can have favorable effects on post-thawed rooster sperm motility parameters, viability, plasma membrane integrity, and abnormality.
