Indian Journal of Animal Research

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Effects of salinity, temperature and pH on the motility in Siganus guttatus spermatozoa

X.R. Huang1, J.Y. Liu1, F. Zhao1, G.P. Feng1, Y. Wang1, T. Zhang1, C. Song1, P. Zhuang1,*
1Laboratory of East China Sea and Oceanic Fishery Resources Exploitation and Utilization, MOA, Shanghai 200090, China.
The effects of salinity, temperature and pH on the activity in the spermatozoa of Siganus guttatus were studied. Milt were collected from 8 wild mature males that came from a commercial hatchery. Sperm motility was measured with a VHS video-recorder and a video camera connected to a microscope. The results showed that most of the sperm were highly motile in 400-700 mM NaCl, the longest duration of sperm motility recorded in 600mM NaCl and the duration was 788.0±16.9 s. Sperm was not motile when suspended at pH 4, sperm motility was observed at pH 5, there was a relatively higher percentage of motile sperm in solutions at pH 5-8 (above 80%), the duration was the longest and achieved (769.0±19.8) s when pH was 8. The motility and duration increased within 10-25ºC and decreased at the range of 25-40ºC, the highest motility and the longest duration were all appeared at 25 ºC, they were (91.0±1.4) % and (725.0±21.2) severally. Optimum salinity, temperature and pH for S.guttatus reproduction was 600mM NaCl, 25 ºC and 8 respectively.
Teleost spermatozoa are immotile while they remain within the spermary. In most instances, they are also motionless in isotonic environment including seminal plasma (Stoss, 1839; Morisawa, 1985). The vitality of motility is inspired by the change of osmotic pressure in the environment (Morisawa and Suzuki, 1980). Sperm activity is an important prerequisite that determine the quality and fertilization ability of sperm (Billard, 1978; Stoss, 1983; Dada, 2012; Frydrychova et al., 2015) and is usually indicated as percentage and duration of active sperm after activation (Lahnsteiner et al., 1996). Some parameters of the motion performance, such as pH, osmotic pressure, ion concentration (Na+, K+, Ca2+, Mg2+), temperature and dilution method, influence the vitality and duration of fish semen (Cosson et al., 1999; Cosson, 2004).

The rabbitfish Siganus guttatus (Bloch 1787), family Siganidae, is widely distributed in the Eastern Indian Ocean and Western Pacific region (Woodland, 1990). All kinds of rabbitfish have been considered as good indicators for aquaculture in the nutrition because of its good viability and low nutrition level in the food chain and most special, because of its wide market cognition (Juario et al., 1985). In China, the artificial breeding and the trophic component of S. guttatus had already been studied (Zhao et al., 2013; Song et al., 2018), but little information is available about the sperm motility of S. guttatus.

In this study, we analyzed the effects of temperature, salinity and pH on the activity and duration of S. guttatus spermatozoa. The purpose of the study is to discuss the effects of some external environmental factors inspiring sperm motion and provide basis and reference during artificial reproduction of S. guttatus.
Eight mature male S. guttatus obtained from a commercial hatchery were reared in indoor tanks at Qionghai Research Center, East China Sea Fishery Research Institute, CAFS, Hainan, China. Semen was gathered with a pasteur pipette and stored in a micro-tube on crushed ice until test within 3 h in the following experiments.

Sperm motility was monitored with a VHS video-recorder linked to a video timer and a video camera connected to a microscope. Spermatozoa were classified as active when, 5s after blending, the sperm head showed forward movement in successive video frames. Observations were made utilizing the “jog-shuttle” function of the video-recorder, which allowed selectable movement of each video frame. Percentage of vitality was determined by evaluating the activity of at least 50 randomly selected spermatozoa for each sample were repeated at least three times and data were averaged.
Experiment 1
The effects of different concentration sodium chloride (NaCl) on the sperm activity and duration were researched. A total of 100 µl of each solution was placed on a glass slide, the sperm was taken by using a clean and dry pasteur pipette, blended with the solution on the glass slide and observed at 20´magnification of an inverted microscope, recorded sperm motility and duration using video-recorder. Samples of repeated experiment came from eight S. guttatus spermatozoa.
Experiment 2
The pH was adjusted by the addition of 0.1 mL HCl or NaOH solutions. The pH of each seawater solution was measured using a pH meter. Activity and duration of sperm was expressed as described in Experiment 1. Samples of repeated experiment came from eight S. guttatus spermatozoa.
Experiment 3
Seven temperature 10°C, 15°C, 20°C, 25°C, 30°C, 35°C and 40°C were chosen in the seawater to activate the sperm. The temperature was controlled by low-constant temperature incubator. The experimental process was similar to Experiment 2. Samples of repeated experiment came from eight S. guttatus spermatozoa.
Statistical analysis
Datas in all experiments were expressed as mean values±S.E.M. The statistical analysis of data was made using analysis of variance, Duncan’s multiple range test (DMRT) was used to examine the significant difference. Significance of results was determined at P<0.05.
Experiment 1
Sperm motility and duration in NaCl solutions of different concentrations are shown in (Table 1). Most of the S. guttatus sperm had higher motility at the range of 400-700 mM NaCl solution, the highest motility reached 91.5±2.1% when NaCl concentration was 600 mM. Duration of sperm movement increased within 250-600 mM NaCl solution, the longest duration achieved 788.0±16.9 s when NaCl concentration was 600 mM.

Table 1: Effects of different concentration of NaCl on the sperm motility and duration.

Experiment 2
The results of effects of different pH solution on sperm activity and duration are shown in (Table 2) Significant test for the effects of pH on the sperm activity and duration are shown in (Table 3 and Table 4). The sperm had no activity at pH 4, sperm activity was observed at pH 5, and there was a higher motility in solutions at the range of pH 5-8 (above 70%) than at other pH tested. Duration of sperm was relatively longer within pH 5-8 (above 400 sec), the longest duration recorded at pH 8 and the time was 769.0±19.8 s.

Table 2: Effects of different concentration of pH on the sperm motility and duration.

Table 3: Significant test for the effects of pH on the sperm motility.

Table 4: Significant test for the effects of pH on the sperm duration.

Experiment 3
Sperm activity and duration in different temperature seawater are shown in (Table 5). Significant test for the effects of temperature on the sperm activity and duration are presented in (Table 6 and Table 7). The activity and duration added at the range of 10-25°C gradually, best activity and the longest duration recorded at 25°C, subsequently, the activity and duration declined significantly with the increase of temperature, the sperm motility and duration dropped to (65.0±7.1) % and 599.5±20.5 s respectively at 40°C.

Table 5: Effects of different temperature on the sperm motility and duration.

Table 6: Significant test for the effects of temperature on the sperm motility.

Table 7: Significant test for the effects of temperature on the sperm duration.

The osmotic concentration of the activator had a significant effect on the activity of sperm that became motile after dilution (Takai and Morisawa, 1995). Obviously, lower than 250 mM NaCl solution was unable to activate sperm of S. guttatus, the sperm was also motionless in more than 850 mM NaCl, the sperm had better motility at the range of 500-600 mM NaCl (salinity 30-35). The optimum salinity required for sperm movement is closely related to the reproductive salinity of the species (Jiang et al., 2000). Current research results were consistent with the salinity required (32.5-34) during reproduction of S. guttatus (Liu et al., 2009). Ohta and Izawa (1996) reported that eel spermatozoa were motionless in 300-400 mOsm/kg NaCl, but the sperm could be activated in more than 500 mOsm/kg solutions, the results didn’t agree with ours. In current study, the sperm could be activated in more than 250 mM (about 500 mOsm/kg) NaCl and the sperm had no motility in 250 mM (about 500 mOsm/kg) NaCl. One factor motivating the activity of the S. guttatus spermatozoa was an osmotic pressure increase, so the diluents of S. guttatus spermatozoa should be isotonicity. Chambeyron and Zohar (1990) also emphasized the importance of isotonicity in the diluents for marine teleost (Sparus aurata) spermatozoa, the motility of spermatozoa was inspired by the increase of osmotic pressure.

Generally speaking, pH has been thought as having no significant effect on the activity of fish spermatozoa. However high pH could enhance and prolong sperm activity in different freshwater and marine teleosts (Stoss and Donaldson, 1982). The spermatozoa of Epiniphelus coioides was more adaptable to pH and the sperm motility was the highest at pH 6.5~8.7 (Zhao et al., 2003). The eel spermatozoa had a wide range of adaptation to pH and had high vitality within the range of pH 4~11, the spermatozoa had higher motility and longer duration at pH 3 and 4, which was very unique (Huang et al., 2011). In current study, seawater of pH 4 and pH 12 couldn’t activate S. guttatus spermatozoa, there was no significant difference in sperm activity at the range of pH 4-12. The results showed that S. guttatus spermatozoa had higher activity and longer duration at pH 5-8, which also implied that S. guttatus spermatozoa could adapt to a limited range of pH.

The velocity, activity and longevity of spermatozoa rely on temperature of the activating solution (Stoss, 1983; Billard et al., 1995). Because the energetic resources of fish spermatozoa are limited, an increase in velocity caused by such a temperature rise of the living water environment leads to reduce the duration of movement (Schlenk and Kahmann, 1938). The studies on the rainbow trout spermatozoa showed that both the motility and the movement duration declined when the temperature of the water environment increased (Billard and Cosson, 1988). The optimum temperature of sperm motility in E. coioides was 25-31°C (Zhao et al., 2003), which was a little higher than that of S. guttatus. The activity and duration of eel spermatozoa added with increase of temperature at the range of 18-24°C, but the activity and duration declined with addition of temperature at the range of 24-30°C (Huang et al., 2011), the results was similar to ours. In present study, the motility and duration of S. guttatus spermatozoa increased at the range of 10-25°C gradually, but the activity and longevity declined markedly at the range of 25-40°C. The results indicated that temperature had great effects on the activity of S. guttatus spermatozoa.
This study was financially supported by grants from Agriculture Commission of Shanghai (project NO. 2016/2-3) and the National Infrastructure of Fishery Germplasm Resources (2018DKA30470).

  1. Billard, R.(1978). Changes in structure and fertilizing ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture.,14: 187-198.

  2. Billard, R., Cosson, M.P.(1988). Sperm motility in Rainbow trout, Parasalmo gairdneri; effects of pH and temperature. In: Reproduction in Fish Basic and Applied Aspects in Endocrinology and Genetics. Breton B, Zohar Y, editors. INRA Paris: 161-167 pp.

  3. Billard, R., Cosson, J., Perchec, G., Linhart, O.(1995). Biology of sperm and artificial reproduction in carp. Aquaculture., 124 b: 95-112.

  4. Chambeyron, F., Zohar, Y.(1990). A diluents for sperm cryopreservation of gilthead seabream Sparus aurata, Aquaculture., 90:345-352.

  5. Cosson, J.(2004). The ionic and osmotic factors controlling motility of fish spermatozoa. Aquacult. Int., 12:69-85.

  6. Cosson, J., Billard, R., Cibert, C., Dreanno, C., Suquet, M.(1999). Ionic factors regulating the motility of fish sperm. In: The Male Gamete: from basic to clinical applications Gagnon C, editor. Cache Rive Press; 161-186 pp.

  7. Dada, A.A.(2012). Effect of ascorbic acid supplementation in broodstock feed on sperm quality of African sharptooth catfish (Clarias gariepinus). Indian J. Anim. Res., 46(3): 213- 218.

  8. Frydrychova, S.,Lustykova, A.,Vaclavkova, E., Lipensky, J.,Rozkot, M.(2015).Effect of different extenders on quality of frozen-thawed boar semen. Indian J. Anim. Res., 49(6): 851- 854.

  9. Huang,X.R., Zhuang, P., Zhang, L.Z., Qiao, Z.G., Jiang, Q., Liu, J.Y., Yao, Z.F., Feng, G.P. (2011). Effects of extracellular environment factors on the motility in Japanese eel spermatozoa. Cell Biology International., 35:505-508.

  10. Jiang,S.G., Li,J.E., Ou,Y.J., Zheng,Y.T.(2000). Relationships between conditions for activating spermatozoa of four sparidae fishes and the fishes’ ecological habits. Acta Ecologica Sinica., 20(3):468-473(in Chinese).

  11. Juario, J.V., Duray, M.N., Duray, V.M., Nacario, J.F., Almendras, J.M.(1985). Breeding and larval rearing of the rabbitfish, Siganus guttatus(Bloch). Aquaculture., 44: 91-101.

  12. Lahnsteiner, F., Berger, B., Weismann, F., Patzner, R.A.(1996). Motility of spermatozoa of Alburnus alburnus (Cyprinidae) and its relationship to seminal plasma composition and sperm metabolism. Fish Physiol. Biochem.,15:167-179.

  13. Liu, J., Zhang, L., Zhuang, P., Zhao, F., Zhang, T., Feng, G., Huang, X., Yan, W., Hou, J.(2009). Study on artificial propagation of Siganus guttatus. Mar Fish.,31:73-81(in Chinese).

  14. Morisawa, M.(1985). Initiation mechanism of sperm motility at spawning in teleosts. Zool. Sci., 2: 605-615.

  15. Morisawa, M., Suzuki, K.(1980). Osmolality and potassium ion; Their roles in initiation of sperm motility in teleosts. Science., 210: 1145-1147.

  16. Ohta, H., Izawa, T.(1996). Diluent for cool storage of the Japanese eel (Anguilla japonica) spermatozoa. Aquaculture., 142:107-118.

  17. Schlenk, W., Kahmann, H.(1938). The chemical composition of seminal fluids and their physiological importance study with trout sperm. Biochemical Zool.,295:283-301.

  18. Song C.,Zhao F., Liu J.Y.,Wang Y.,Huang X.R.,Zhuang P.(2018). Proximate composition and fatty acid profile in different tissues of juvenile Siganus guttatus fed with Enteromorpha pfolifera. Indian J. Anim. Res., DOI:10.18805/ijar. B-886.

  19. Stoss, J.(1983). Fish gamete preservation and spermatozoa physiology. In: Hoar, W. S., Randall, D. J., Donaldson, Fish Physiology, E. M., (Eds.), Part B, vol. 9. Academic Press, New York; 305-350pp.

  20. Stoss, J., Donaldson, E.M.(1982). Preservation of fish gametes. In:, International Symp. Reprod. Physiol. Fish, Goos, H. J. Th., Richter, C. J. J., (Compilers). Wageningen, The Netherlands, 114-122pp.

  21. Takai, H., Morisawa, M.(1995). Change in intracellular K+ concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts. J. Cell. Sci., 108:1175-1181.

  22. Woodland, D.J. (1990). Revision of the fish family Siganidae with descriptions of two new species and comments on distribution and biology. Indo-Pac Fish., 19: 1-136.

  23. Zhao, F., Wang, Y., Zhang, L.Z., Zhuang, P., Liu, J.Y.(2013). Survival, growth, food conversion efficiency and plasma osmolality of juvenile Siganus guttatus (Bloch, 1787): experimental analyses of salinity effects. Fish Physiol Biochem., 39:1025-1030.

  24. Zhao, H.H., Liu, X.C., Lin, H.R., LiuFu, Y.Z.,Wang,Y.X.(2003). Ultrastructure of spermatozoa and effects of salinity, temperature and pH on spermatozoa motility in Epiniphelus coioides. Journal of Fishery Sciences of China., 10(4):286-292(in Chinese). 

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