| This article utilized microscope and flow cytometry (FCM) after eosin or propidium iodide(PI) stain to study the feasibility of FCM in the spermatozoa quality evaluation, cryopreservation techniques and the mechanism of freezing injury of the Coelomactra antiquata's spermatozoa. The aim was to build a mature cryopreservation of the C. antiquata's spermatozoa and to provide scientific basis and technical support for artificial breeding study and genetics breeding study of C.antiquata. The main results were as follows:In order to evaluate the feasibility of FCM used for detecting the survival rate of spermatozoa of C. antiquata, this article utilized eosin staining and PI staining with optical microscope and FCM to study the correlation and precision between them. The results showed that there was positive correlation between eosin and PI staining(r=0.954, P=0.003), that was these stainings could be used as effective methods to detect the spermatozoa survival rate of C. antiquata. But the precision of PI staining (average CV was 2.39%) was better than eosin's (average CV was 4.67%). Therefore FCM was an effective method to detect the survival rate of the spermatozoa of C. antiquata.This paper used spermatozoa survival rate as evaluation index and utilized FCM to study the effect of different cryoprotectants pre-freezing rate and freezing rate on the cryopreservation of C. antiquata spermatozoa. With a certain cryopreservation protocol, we studied dimethylsulfoxide(DMSO), ethylene glycol,1-2 propylene glycol at 1%,2.5%, 4%,5%,10%,15%,20% concentration. Only 4%,10%DMSO,20% PG and EG at 2.5%,5% and 15% were chosen for the following pre-freezing step. Five pre-freezing rates were analyzed:-1℃/min,-4℃/min,-6℃/min,-8℃/min, 10min at 20±1℃, and -4℃/min was chosen for the following freezing procedure among five gradients those were-0.25℃/min,-0.5℃/min,-1℃/min,-4℃/min and plunged into liquid nitrogen(LN2) directly. Thawed spermatozoa were thawed at 35℃water bath, analyzed of the survival rate by FCM and verified with artificial fertilization, the results showed that the survival rate and fertility rate were the best when thawed spermatozoa were cryopreserved by 15%EG,-4℃/min of pre-freezing and -4℃/min of freezing, they were 95.53% and 52.32%, respectively. In order to assess the effect of different cryoprotectants on the quality of thawed spermatozoa, and then study the mechanism of freezing injury of the C. antiquata's spermatozoa, DMSO, EG and PG were tested by FCM on the membrane integrity, apoptosis and mitochondrial activity of the thawed spermatozoa. The results showed that these cryoprotectants all could better maintain the membrane integrity and to some extent promote the apoptosis process of thaw spermatozoa. Moreover, the effect of different cryoprotectant and different concentration of the same cryoprotectant were different on the membrane integrity and apoptosis of thaw spermatozoa,15%EG had the highest rate of membrane integrity and the lowest rate of apoptosis,80.54% and 7.99%, respectively. In addition, the cryopreservation progress made lower of the mitochondrial activity of the thawed spermatozoa. These cryoprotectants all could better protect the mitochondrial activity and 15%EG had the lowest rate of necrosis spermatozoa and the highest rate of active-mitochondrial spermatozoa,15.04% and 81.32%, respectively.Scanning electron microscopy and transmission electron microscopy were used to investigate the variation of morphology and ultrastructure between fresh and frozen-thawed spermatozoa and integrated with the results of FCM in order to study the mechanism of freezing injury of the C. antiquata's spermatozoa. The results showed that the injury of acrosome and mitochondrial was much serious under scanning electron microscopy. The surface of frozen-thawed spermatozoa head presented irregular hollow and the flagellum presented rupture or off and so on. Under transmission electron microscopy, the plasma membrane of frozen-thawed spermatozoa expanded or bursted, the membrane of acrosome fell off, bursted. The membrane system of mitochondrial also were damaged, such as mitochondrial disintegrated, presented vacuolization and so on. These injury performances were the same with the results of FCM which studied the effect of cryopreservation on the membrane integrity, apoptosis and mitochondrial activity of the thawed spermatozoa. So we can say that cryopreservation process really caused certain degrees injury on the plasma membrane, acrosome and mitochondrial of the spermatozoa of C. antiquata. |
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