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

栉江瑶(Atrina pectinata)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，本研究探究了人工海水中铵离子浓度和pH的变化对栉江瑶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)、平均路径运动速率(VAP)和鞭毛摆动频率(BCF)的运动学特征，并对精子ATP含量、ATP酶与超氧化物歧化酶(SOD)活性进行了记录。结果显示，简单提高海水pH可略微提高栉江瑶精子运动率，但无法使精子进入高度活化状态；含有铵离子的碱化海水可有效激发精子游泳运动，3 mmol/L氨海水激活效果最佳。经3 mmol/L氨海水激活后，精子活力等级(MI)在21 min内一直保持在≥4的状态，在激活的前3 min内精子运动率都在80%以上，VCL>56 μm/s，VSL>17 μm/s，VAP>30 μm/s，BCF>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/g prot]，随后，无显著变化(P>0.05)。精子Na+-K+-ATP酶和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±0.03) U/mg prot]，Ca2+-Mg2+-ATP酶活性较高[(6.08± 0.04) U/mg prot]。精子SOD活性在15 min内逐步降低至[(1.23±0.73) U/mg prot]，随后维持稳定。本研究可为栉江瑶精子激活机制深入研究提供基础，助力发展栉江瑶人工繁育技术。

Comparing the effects of ammonia ion concentration and pH on Atrina pectinata sperm activation in artificial seawater

Pen shell (Atrina pectinata) is a Bivalvia species with high commercial value in China. However, large scale production is difficult owing to the lack of seed. Pen shell uses external fertilization with sperm stored in the testis in a nonmotile state. Sperm motility is initiated when they are released from the reproductive tract into the aquatic environment, which enables fertilization. Various chemical signals including pH, ions, and cyclic nucleotides control sperm motility. However, there are interspecific differences in activation conditions between different Bivalvia species. Screening out an effective activating medium is the basis of artificial breeding technology.Swimming parameters, such as total motile sperm (TM), movement velocity, and beat-cross frequency (BCF) are common indicators to evaluate sperm quality. Sperm must have sufficient motility to reach the egg to complete fertilization. In addition, sperm needs a large amount of ATP to maintain its swimming movement. Intracellular ATP content controls the duration of the sperm movement phase in most marine species. Studying the movement characteristics and energy metabolism of sperm during activation will help develop and optimize artificial breeding technology. Studies of A. pectinata mainly focus on their oocytes. However, the activation conditions, moving characteristics, and energy metabolism of sperm during activation remains unknown. Thus, there is an urgent need to screen appropriate media and study the activation mechanisms of A. pectinata sperm. Adult A. pectinata were collected from Wuzhizhou Island, Hainan Province in November 2021. Artificial seawater with different levels of ammonia ions and pH were used to activate sperm. This study examined A. pectinata sperm activation in artificial seawater by varying the ammonia ion concentration and pH. The change in sperm motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), and BCF were described. The ATP content, ATPase activities, and superoxide dismutase (SOD) activity were quantitatively recorded during the full activated stage. The motility was slightly improved by increasing the pH of seawater, but could not achieve the fully activated stage. Furthermore, the motility was significantly improved when activated by alkalized seawater containing ammonia ions, and the best results were observed in groups containing 3 mmol/L ammonia ion: The sperm motility index (MI) was in the fully activated stage (MI ≥ 4) until the end of the experiment, with TM ≥ 80%, VCL > 56 μm/s, VSL > 17 μm/s, VAP > 30 μm/s, and BCF > 6 Hz. Sperm ATP content decreased to 30.29% of their initial values [(128.80±66.92) μmol/g prot] during 5 min post activation and was maintained at this level during post-activation. ATPase activities were maintained at a constant level. Na+-K+-ATPase activity was lower [(0.62±0.03) U/mg prot] compared to Ca2+-Mg2+-ATPase activity [(6.08±0.04) U/mg prot]. The SOD activity of sperm steadily decreased to [(1.23±0.73) U/mg prot] during 15 min post activation and remained stable. In conclusion, pH was not the decisive factor in pen shell sperm motility. Instead, ammonium ion promoted sperm activation. There was a significant decrease in sperm ATP concentration (P<0.05) at the beginning of the post-activation stage and the ATP concentration followed by stabilization at a lower level. The reduction in SOD activity may cause oxidative stress. The findings in this study can be instructive to conduct further research on sperm activation mechanisms, and help develop artificial breeding technology for A. pectinata.