Comparing the Effects of Ammonia Ion Concentration and pH on Atrina pectinata Sperm Activation in Artificial Seawater

Abstract

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.

Keywords

• atrina pectinata
• sperm
• motility
• ammonia ion
• ph
• energy metabolism