STUDY ON ULTRASTRUCTURE OBSERVATION OF FRESH AND CRYOPRESERVATED SPERM IN CRASSOSTREA GIGAS
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摘要: 采用程序降温仪分步降温冷冻保存长牡蛎（Crassostrea gigas）精液，并用扫描电镜、透射电镜研究了精子的超微结构损伤。超低温冷冻保存后长牡蛎精子的运动率、受精率及孵化率与鲜精无显著差异。鲜精中84.5%的精子形态结构正常，冻精中73%的精子形态结构正常。形态结构正常的精子表现为顶体、质膜、线粒体与鞭毛结构完整、染色质形状规则，顶体、线粒体及中心粒结构正常，鞭毛形态完整、微管结构清晰；形态结构异常的精子表现为顶体脱落、解体，精子头部质膜膨胀、破裂、染色质肿胀、破裂、解体，线粒体移位、脱落、膨胀，嵴退化或消失，鞭毛弯折、断裂，微管解聚。结果显示，以10% DMSO为抗冻保护剂，HBSS溶液为稀释液，1：4的稀释比例，添加海藻糖，采用分步降温法冷冻保存，对长牡蛎精子具有较好的抗冻保护作用，合适的冻存方法可以有效的保护太平洋牡蛎精子冷冻过程中结构损伤。研究有助于长牡蛎种质资源的收集保存及应用。Abstract: A step-wise cooling schemes was employed to cryopreserve Crassostrea gigas sperm, and the sperm ultrastructure was observed by scanning electron microscopy and transmission electron microscopy. The results showed that there were no significant differences between frozen-thawed sperm and fresh sperm in the motility, fertilization rate and hatching rate. Both the fresh sperm and cryopreserved sperm had ultrastructural damages. The normal rates of the fresh and cryopreservated sperms were 84.5% and 73%, respectively. The cryopreserved sperm without damage had normal morphology in the plasma membrane, mitochondria and nuclear, the acrosome, and centriole, and the mitochondrion obtained integrity with well-developed cristae. The sperm cryodamages with damages had swelled or disrupted plasma and nuclear membrane, partially damaged nucleus and swelled, dislocated or disarticulated mitochondrion with degenerated or vanished cristae. The results showed that HBSS with at 1:4 dilution trehalose and 10% DMSO is the best condition for extender and cryoprotecant and for protecting the frozen-thawed C. gigas sperm, which will benefit the preservation of C. gigas and application of sperm cryopreservation skills.
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Keywords:
- Crassostrea gigas /
- Sperm /
- Cryopreservation /
- Vitality /
- Ultrastructure
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图 1 长牡蛎鲜精扫描电镜观察
A.鲜精整体扫描电镜照片; B、D.单个精子扫描电镜图, 示精子头部近似圆形, 顶体位于头部前方, 部分线粒体整齐排列, 鞭毛细长; C.示精子头部结构, 顶体、头部、线粒体及鞭毛依次排列; h:头; t:鞭毛; a:顶体; mi:线粒体
Figure 1. The ultrastructure of fresh spermatozoa ofCrassostrea gigas by SEM
A. Fresh sperm of SEM; B, D. Single sperm of SEM, The sperm head approximate circular, acrosome located in the front of head, Some mitochondria ranked neatly, flagella slender; C. The structure of sperm head, head, acrosome mitochondria and flagella lined. h: head; t: flagellum; a: acrosome; mi: mitochondria
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图 2 长牡蛎冻精扫描电镜观察
A.冻精整体扫描电镜照片, 示鞭毛脱落、折断; B.两个精子扫描电镜图, 示头部顶体脱落, 质膜皱缩变形; C、D.单个精子扫描电镜图, 示部分线粒体脱落、破损, 质膜皱缩, 胞膜冻裂, 细胞液外流; h:头; a:顶体; t:鞭毛; mi:线粒体
Figure 2. The ultrastructure of cryopreserved spermatozoa ofCras-sostrea gigas by SEM
A. The whole frozen sperm of SEM, flagellum fall off, bend; B. Two frozen sperm of SEM, acrosome fall off head, plasmalemma collapse; C, D. Single frozen sperm of SEM, some of mitochon dria fall off, damaged, plasmalemma plasmolysis, cell membrane frost cleft, cytosol outflow; h: head; a: acrosome; t: flagellum; mi: mitochondria
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图 3 长牡蛎鲜精透射电镜观察
A.鲜精纵切, 示单个精子整体纵切; B.鲜精头部纵切, 示鲜精头部超微结构; C.鲜精横切, 示线粒体超微结构; D.鲜精鞭毛横切, 示鞭毛典型“9+2”结构; a:顶体; b:亚顶体腔; c:中心体; t:鞭毛; cm:中央微管; dm:双连体; pm:质膜; mi:线粒体; nu:核
Figure 3. The ultrastructure of fresh spermatozoa ofCrassostrea gigas by TEM
A. Fresh sperm rip cutting; B. Fresh sperm head rip cutting, show ultrastructure; C. Fresh sperm rip cutting, show mitochondria ultrastructure; D. Fresh sperm flagellum crosscut, show "9+2" structure. a: acrosome; b: cavity; c: centrosome; t: flagellum; cm: central microtubules; dm: double conjoined; pm: plasma membrane; mi: mitochondria; nu: nucleus
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图 4 长牡蛎冻精透射电镜观察
A.冻精纵切, 示胞膜破裂, 染色质破损、变形, 鞭毛脱落; B.冻精斜切, 示质膜肿胀, 顶体解体, 线粒体损伤; C.鲜精纵切, 示线粒体脱落, 染色质破损; D.冻精鞭毛横切, 下部鞭毛示精子典型“9+2”, 上部鞭毛示微管解聚, 膜皱缩变形; a:顶体; b:亚顶体腔; c:中心体; t:鞭毛; cm:中央微管; dm:双连体; pm:质膜; mi:线粒体; nu:核
Figure 4. The ultrastructure of fresh spermatozoa ofCrassostrea gigas by TEM
A. Frozen sperm rip cutting show membrane rupture, chromatin damaged, flagellum fall off; B. Frozen sperm bevel, show plasma membrane swollen, disintegration of acrosome, mitochondria damage; C. Frozen sperm rip cutting, show mitochondrial loss, chromatin damaged; D. Frozen sperm flagellum crosscut, show "9+2" structure. a: acrosome; b: cavity; c: centrosome; t: flagellum; cm: central microtubules; dm: double conjoined; pm: plasma membrane; mi: mitochondria; nu: nucleus
表 1 长牡蛎鲜精及冻精的运动率、受精率及孵化率(n=3, X±SD)
Table 1 Motility, fertilization rate and hatching rate of fresh sperm and frozen-thawed sperm ofCrassostrea gigas (n=3, X±SD)
精子种类
Sperm运动率
Motility (%)受精率
Fertilization
rate (%)孵化率
Hatching
rate (%)鲜精
Fresh sperm82.23±4.67a 94.90±0.95b 88.89±15.16c 冻精
Frozen sperm71.53±1.00a 95.04±1.99b 93.33±13.33c 
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