EFFECT OF CATION CONCENTRATIONS (K +, Ca 2+ ) AND HORMONAL STIMULATION ON SPERM MOTILITY OF COMMON CARP Cyprinus carpio

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1 EFFECT OF CATION CONCENTRATIONS (K +, Ca 2+ ) AND HORMONAL STIMULATION ON SPERM MOTILITY OF COMMON CARP Cyprinus carpio ABTRACT Received: 25/1/2016; Revised: 08/7/2016; Accepted: 26/9/2016 Le Minh Hoang 1, Pham Phuong Linh 1 Semen quality assessment through sperm motility plays an important role in the production of artificial fi sh and contributes to the preservation of fi sh sperm more effectively. This study aims to evaluate the effect of cation concentrations and hormonal stimulation on sperm motility of common carp. Experiments on the effect of cation concentrations use two types of cations in the following concentrations: Ca 2+ (0.5; 2.5; 5 mm) and K + (5; 25; 50 mm), then use the best cation concentration to check the effects of the injection of the hormone on sperm motility. The results showed that the best cation concentration was 50 mm (K + ) and 2.5 mm (Ca 2+ ). These parameters before and after hormonal stimulation showed no difference. The results of this experiment can help us to create an environment for carp sperm activity based on the above parameters. Keywords: Carp, Cyprinus carpio, cation, hormone stimulation I. INTRODUCTION Spermatozoa of both marine and freshwater fi sh species are immotile in the testis and seminal plasma. Motility of spermatozoa occurs after they are released into surrounding aqueous environment during natural reproduction or into a diluents artifi cial reproduction (Islam and Akhter 2011). Spermatozoa motility is a prerequisite parameter in determining fi sh semen quality and fertilizing capacity (Le et al. 2011). However, spermatozoa motility is also infl uenced by several factors such as: temperature, dilution ratio, different diluents, ion concentration or osmolality (Le et al. 2011) and time after hormonal stimulation (Kowalski et al. 2012). Some studies have carried out with Persian surgeon - Acipenser persicus (Alavi et al. 2004), European perch - Perca fl uviatilis (Alavi et al. 2007), yellow croaker - Larimichthys polyactis (Le et al. 2011), tiger grouper - Epinephelus fuscoguttatus (Hoang and Le, 2014). Le and Hoang (2015) reported the effects of diluent ratio, diluents and osmolality on sperm motility of common carp. Up to now, there are no studies those related to the effect of cation concentration and hormonal stimulation on sperm motility have been done in common carp spermatozoa. Common carp is traditional species of Vietnam. It distributes in ponds, lakes, from Northern to Southern. Besides, it has high commercial value on the market. Nowadays, the indiscriminate exploitation of the wild with the introduction of new multi-line carp gradually causes the loss of pure carp seed. The determination of the optimal environmental factors for sperm motility of common carp has an important role in enhancing the rate of fertilization as well as helps preserve carp sperm more effectively so that we will preserve pure carp seed varieties to cater for the breeding and protection of genentic resources. However, there is no research or publication on the effect of cation concentration and hormonal stimulation on sperm motility of common carp in Vietnam. Therefore, this study aims to evaluate the 1 Institute of Aquaculture, Nha Trang University (NTU), Vietnam NHA TRANG UNIVERSITY 17

2 effect of cation concentrations and hormonal stimulation on sperm motility of common carp. II. MATERIALS AND METHODS All experiments were carried out at the Institute of Aquaculture in Nha Trang University. Male carp were collected from the wild and then semen were stripped in 1.5 ml eppendofe and held on ice and immediately brought to the laboratory for observation and analysis. Spermatozoa motility (SM) assessment: SM was determined immedicately after semen collection. The percentage of sperm exhibiting rapid, vigorous and forward movement were classifi ed under a microscope by diluting the sperm into distilled water at ratio 1:100 (mixing 1µl semen and 99 µl distilled water). * Effect of cation concentrations on SM: To assess the effect of cation concentrations on motility, semen was diluted at ratio 1:25 in cation solution concentrantions: KCl (5 mm, 25 mm or 50 mm) and CaCl 2 (0.5 mm, 2.5 mm, 5mM). Then, check SM and analyse this results to fi nd the optimal cation concentration. * Effect of injection hormone on SM: All treatments of effect of injection hormone on motility were conducted similar to the above experiments. Males were injected with hormone LHRHa. Then semen was stripped after 6 hours and 12 hours. These sperm were tested by the best dilution ratio 1:25, the best diluents as 0.3% NaCl, the best osmolality at 100 mosm/kg (Le and Hoang, 2015) and the best ion Ca 2+ and ion K + (in this study). Data representing infl uence of dilution ratio, diluents and cation concentration on motility were analyzed by one-way ANOVA using SPSS Results are presented as mean±standard error. Difference with a probability value (P) of 0.05 (P<0.05) were considered signifi cant. III. RESULT AND DISCUSSION 1. Effect of K + and Ca 2+ concentrations on sperm motility 1.1. Ion K + Effect of the change of ions and osmotic pressure level of ambient environment on SM has demonstrated by Morisawa (Seifi et al. 2011) and they have considered as two factor of infl uence SM. In family carp, ion K + may increase velocity and motile sperm so that channel K + has capacity inhibited movement of sperm fl agellum (Islam and Akhter 2011). Effect of K + concentration was described in Figure 1. Figure 1. Effect of K + concentrations on sperm motility of common carp. Values with different alphabetic letters on each bar indicate significant difference between concentrations of K + (P<0.05) 18 NHA TRANG UNIVERSITY

3 The results showed in Figure 1 indicated that the best concentration of K + is 50 mm with time and percentage motility is 835.5±292.2 s and 92.5±1.1%, respectively. No signifi cant difference between concentrations of 5, 25 or 50 mm, however, time motility at 5 and 25 mm is lower than 50 mm. Alavi et al (Alavi and Cosson 2006) also indicated the same result that solution containing mm KCl is able to stimulate SM effectively. Increasing the concentration of K + of solution also increase velocity of SM in European perch - Perca fl uviatilis and the best SM has observed at 50 mm K + but ion K + has no signifi cant effect on the proportion SM. In general, the highest percentage motility has observed at concentration of K + above 20 mm when measured within 15 s after activation (Alavi et al. 2007). For yellow croaker - Larimichthys polyactis, the best SM at concentration of 0.4 M (Le et al. 2011). Therefore, the concentration of K + is different on each fi sh species Ion Ca 2+ Sperm motility can be initiated by alteration of the concentration of Ca 2+ ions in many species, such as in cyprinids (Islam and Akhter 2011). The presence of extracellular Ca 2+ is necessary for the initiation of SM in carp (Krasznai et al. 2000). Effect of concentrations Ca 2+ on SM in carp was described in Figure 2. Figure 2. Effect of Ca 2+ concentrations on sperm motility of common carp. Values with different alphabetic letters on each bar indicate significant difference between concentrations of Ca 2+ (P<0.05) The result showed that the optimal concentration of Ca 2+ in carp sperm is 2.5 mm with time and percentage motility is 51±0.5 s and 86.6±2.5%, respectively. At concentrations of 0.5 or 5 mm, motile time is 46.3±1.7 s and 40.8±2.4 s. No signifi cant difference between concentrations of Ca 2+. Some studies have indicated that the role of concentration Ca 2+ in increasing sperm motility patameters includes time total motility, percentage motility and velocity of sperm. Studying on European perch-perca fl uviatilis indicated that the concentration of Ca 2+ at 2.5 mm increased motile sperm percentage (Alavi et al. 2007). For yellow croaker-larimichthys polyactis, the highest motile sperm obtained in solution containing 0.2 M CaCl 2 (Le et al. 2011). When extracellular Ca 2+ enters into the cell membrane of sperm, it will increase the concentration of intracellular Ca 2+ and stimulate sperm motility. Therefore, Ca 2+ is an important ion to stimulate motile sperm. 2. Effect of hormonal stimulation on sperm motility Using hormone to stimulate maturation of gonad fi sh is a popular factor in aquaculture (Zohar and Mylonas 2001). Wild carp stimulation to produce of milt in captivily requires hormonal (Seifi et al. 2011). Comparing effect of injecting hormone on SM was described in Figure 3. NHA TRANG UNIVERSITY 19

4 Figure 3. Sperm motility in relation time of hormonal stimulation. Different alphabetic letters on each bar indicate significant difference (P<0.05) The activation medium as NaCl 0.3% was used to analyse the motile sperm before and after hormone injection. After analyzing, the result showed that there is no signifi cant difference (P>0.05). At concentration of 100 mosm/kg, 24 hours after injection, motile sperm increased slightly but not with signifi cant difference (P>0.05) for both K + and Ca 2+ ions. Studying on other species, Krol et al (Krol et al. 2009) observed time of motile sperm in European smelt-osmerus eperlanus increase when stimulating by ovaprim+domperidone. For European eel-anguilla Anguilla, injection of HCG does not effect SM (Austriano et al. 2006). Frequently, using hormone to stimulate releasion of sperm in carp does not effect sperm motility. IV. CONCLUSION AND RECOMMENDATION - The optimal ion K + concentration was 50 mm with time and percentage motility 835.5±292.2 s and 92.5±1.1%, respectively - The optimal ion Ca 2+ concentration was 2.5 mm with motile time at 51±0.5 s and percentage of motile sperm at 86.6±2.5%. - Be able to use NaCl 0.3%, medium 100 mosm/kg, medium KCl 50 mm and CaCl mm for evaluating sperm motility but these parameters before and after hormonal stimulation show no difference. - Future research should use another hormones and fertility test for evaluating sperm motility. REFERENCES 1. Alavi S.M.H. and C osson J., Sperm motility in fishes. (II) Effects of ions and osmolality: A review. Cell Biology International, 30: Alavi S.M.H., Coss on J., Karami M., Amiri M.B. and Akhoundzadeh M.A., Spermatozoa motility in the Persian sturgeon, Acipenser persicus: effects of ph, dilution rate, ions and osmolality. Reproduction, 128: NHA TRANG UNIVERSITY

5 3. Alavi S.M.H., Rodi na M., Policar T., Kozak P., Psenicka M. and Linhart O., Semen of Perca fluviatilis L.: Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility. Theriogenology, 68: Austriano J.F., Mr co-jimenez F., Perez L., Balasch S., Garzon Penaranda D.L., Vicente D.S., Viudes-de-Castro J.S. and Jover M., Effect of HCG as spermiation inducer on European eel semen quality. Theriogenology, 66: Bastami K.D., Iman pour M.R. and Hoseinifar S.H., Sperm of feral carp Cyprinus carpio: optimization of activation solution. Aquaculture International, 18: Cabrita E., Robles V. and Herráez P., Sperm quality assessment. In: E. Cabrit, V. Robles, and P. Herráe (eds). Methods in Reproductive Aquaculture Marine and Freshwater Species: CRC Press Taylor & Francis Group: Cosson J., Groison L.A., Suquet M., Fauvel C., Dreanno C. and Billard R., Marine fish spermatozoa: racing ephemeral swimmers. Reproduction September 1, 136: Islam S.M. and Akhte r T., Tale of fish sperm and factors affecting Sperm Motility: A Review. Advances in Life Sciences, 1: Krasznai Z., Marian T., Izumi H., Damjanovich S., Balkay L., Tron J. and Morisawa M., Membrane hyperpolarization removes inactivation of Ca2 + channels, leading to Ca2 + influx and subsequent initiation of sperm motility in the common carp. Proc. Natl Acad Sci USA, Krol J., Kowalski R. K., Hliwa A., Dietrich G.J., Stabinski R. and Ciereszko A., The effects of commercial preparations containing two different GnRH analogues and dopamine antagonists on spermiation and sperm characteristics in the European smelt Osmerus eperlanus. Aquaculture International, 286: Kowalski R.K., Hliwa P., Cejko B.I., Krol J., Stabinski R. and Ciereszko A., Quality and quantity of smelt (Osmerus eperlanus) sperm in relation to time after hormonal stimulation. Biology of Reproduction, 12: Lahnsteiner F., Berg er B., Weismann T. and Patzner A.R., Sperm structure and motility of the freshwater teleost Cottus gobio. Journal of Fish Biology, 50: Le M.H., Lim H.K., M in B.H., Park M.S., Son M.H., Lee J.U. and Chang Y.J., Effects of varying dilutions, ph, temperature and cations on spermatozoa motility in fish Larimichthys polyactis. Environmental Biology, 32: Le M.H. and Hoang H.G., Effect of dilutions ratio, diluents and osmolality on sperm motility in common carp Cyprinus carpio. Journal of Fisheries Science and Technology, 4: Seifi T., Imanpoor R.M. and Golpour A., The Effect of Different Hormonal Treatments on Semen Quality Parameters in Cultured and Wild Carp. Turkish Journal of Fisheries and Aquatic Sciences, 11: Zohar Y. and Mylonas C.C., Endocrine manipulation of spawning induction in cultured fish from hormone to gene. Aquaculture International, 197: NHA TRANG UNIVERSITY 21