文章摘要
不同铵离子浓度与pH人工海水对栉江瑶精子激活效果的比较
Comparison of the effects of ammonia ion concentration and pH in artificial seawater to Atrina pectinata sperm activation
投稿时间:2023-01-20  修订日期:2023-02-19
DOI:
中文关键词: 栉江瑶  精子  运动率  离子氨  pH  能量代谢
英文关键词: Atrina pectinata  sperm  motility  ammonia ion  pH  energy metabolism
基金项目:基金项目: 海南省自然科学青年基金(320QN207);海南大学科研启动基金(KYQD(ZR)20061);海南省海南省院士创新平台建设项目(HD-YSZX-202011)
作者单位邮编
邱达观 海南大学海洋学院 570228
兰帅勤 海南大学海洋学院 
陈煜 海南大学海洋学院 
毛晶 海南大学海洋学院 
臧战 海南大学海洋学院 
VASQUEZ Hebert Ely 海南大学海洋学院 
於锋 海南大学海洋学院 
王爱民 海南大学海洋学院 
郑兴* 海南大学海洋学院 570228
顾志峰 海南大学海洋学院 
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中文摘要:
      栉江瑶为我国重要的海洋经济双壳贝类,近年来其养殖生产活动备受养殖企业关注,为发展和优化其人工繁殖技术,本文探究了人工海水中铵离子浓度和pH的变化对新鲜精子游泳运动的激活作用,定量描述了高度活化状态下精子曲线运动速率 (VCL)、直线运动速率(VSL)、平均路径速度(VAP)、鞭毛摆动频率(BCF)的运动学特征,并对精子ATP含量、ATP酶与SOD酶活性进行了记录。结果显示:简单提高海水pH可略微提高精子运动率,但无法使精子进入高度活化状态;有铵离子存在的碱化海水可有效激发精子游泳运动,3 mM氨海水激活效果最佳。经3 mM氨海水激活后,精子活力等级在21 min内一直保持在MI ≥ 4的状态,在激活的前3 min内精子运动率都在80%以上,VCL大于56 μm/s,VSL大于17 μm/s,VAP大于30 μm/s,BCF大于6 Hz。精子ATP含量在激活5 min后降低至初始含量的30.29% (128.80 ± 66.92 μmol·gprot-1),随后无显著变化(P>0.05)。精子Na+K+-ATP酶活性与Ca2+Mg2+-ATP酶活性在运动过程中较为稳定,其中Na+K+-ATP酶活性较低 (0.62±0.029 μmolPi/mgprot/hour),Ca2+Mg2+-ATP酶活性较高 (6.08±0.036 μmolPi/mgprot/hour)。精子SOD酶活性15 min内呈逐步降低趋势,随后维持稳定。本研究可为栉江瑶精子激活机制深入研究基础,助力发展栉江瑶人工繁育技术。
英文摘要:
      The pen shell (Atrina pectinata) is one of bivalvia species with high commercial value in China, of which the production is difficult in large scale due to lacking enough seed. For external fertilization species like pen shell, sperm stored in testis in a nonmotile state. Sperm motility is initiated when they are released from the reproductive tract into the aquatic environment, which make them have fertilization ability. Various chemical signals including pH, ions and cyclic nucleotides are involved in controlling sperm motility. However, there are interspecific differences of activation conditions between different bivalvia species. Screening out effective activating medium is the basis of artificial breeding technology. Motion parameters such as total motile sperm (TM), movement velocity, Beat-cross frequency (BCF) are common indicators to evaluate sperm quality. Sperm must have enough motility to reach the egg to complete fertilization. In addition, sperm needs a large amount of ATP to maintain its swimming movement. In most marine species, intracellular ATP content controls the duration of sperm movement phase. Study on the movement characteristics and energy metabolism of sperm during activation will help to develop and optimize artificial breeding technology. However, the study of A.pectinata mainly focus on their oocytes. Research towards sperm motivation is very limited. The activation conditions, moving characteristics and energy metabolism of sperm during activation still unknown. Thus, there is an urgent need to screen appropriate medium and study the activation mechanisms of A.pectinata sperm. In November 2021, adult A.pectinata were collected from Wuzhizhou Island, Hainan Province. Artificial seawater with different levels of ammonia ion and pH were used to motivate sperm. The effects of ammonia ion concentration and pH in artificial seawater to A. pectinata sperm activation were conducted in present study. The change of sperm motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), beat-cross frequency were described using computer-aided sperm analysis(CASA) system. The ATP content, ATPase activities and SOD activity during the full activated stage were recorded quantitatively. The results are shown as follows: the motility can be slightly improved by increasing the pH of seawater, but could not arrived the full activated stage. Furthermore, the motility could be improved significantly when activated by alkalized seawater containing ammonia ion, and the best results were observed in groups containing 3 mM ammonia ion. After 3 mM ammonia seawater activated, the sperm motility index (MI) were in full activated stage (MI ≥ 4) until the end of the experiment, where the total motile sperm (TM) ≥ 80%, VCL> 56 μm/s, VSL > 17 μm/s, VAP > 30 μm/s and BCF > 6 Hz were observed. Sperm ATP content decreased to 30.29% of their initial values during 5 mins post activation and was maintained at this level during post-activation stage. ATPase activities maintained at a constant level. Na+K+-ATPase had a lower activity (0.62±0.029 μmolPi/mgprot/hour) compared to Ca2+Mg2+-ATPase (6.08±0.036 μmolPi/mgprot/hour). SOD activity of sperm decreased steadily to 34.08%(1.23 ± 0.73 U/mgprot) of initial values during 15 mins post activation and remained stable. In conclusion, the results of the present study indicate that pH is not the decisive factors of pen shell sperm motility. Ammonia ion had a promotion effect on sperm motivation. There was a significant decrease in sperm ATP concentration (P<0.05) at the beginning of post-activation stage and then ATP concentration maintained stable. Although ATPase activities kept steady during the experiment, the reduction of SOD activity may cause sperm under the risk of oxidative stress. The finding in present study can be instructive to further research on sperm activation mechanisms, and helps to develop artificial breeding technology for Atrina pectinata.
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