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. |