Saeed Hajirezaee, Bagher Mojazi Amiri, Ali Reza Mirvaghefi

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1 Research Journal of Fisheries and Hydrobiology, 5(1): 27-31, , INSInet Publication Relationships Between the Chemical Properties of Seminal Fluid and the Sperm Motility Characteristics of Caspian Brown Trout, Salmo Trutta Caspius (A Critically Endangered Salmonid Fish) Saeed Hajirezaee, Bagher Mojazi Amiri, Ali Reza Mirvaghefi Department of Fisheries and Environmental Sciences, Faculty of Natural Resources, University of Tehran, Karaj, Iran. Abstract: In this study, the relationships between the chemical properties of seminal fluid and sperm motility characteristics (percentage and duration of sperm motility) of Caspian brown trout, Salmo trutta caspius were investigated. In this regard, significant positive relationships were found between the duration of spermatozoa motility and ph of semen, also, the percentage of motile spermatozoa and the - concentrations of Na, Cl, K, Ca, Mg and osmolality of semen. The understanding of such relationships could be useful for formulating of species-specific diluting media (Extender) for dilution of semen of this species during the cryoperservation of semen. Key words: Chemical properties of seminal fluid, sperm motility, dilution, Cryopreservation, Caspian brown trout INTRODUCTION Because of chemical properties of seminal fluid, fish spermatozoa are immotile in seminal fluid. Immotile status is necessary to maintain the potential of sperm motility until sperm ejaculation to spawning medium i.e. water. When sperm is released to water, the dilution of semen in water stimulates the simultaneous activation of sperm cells and fertilization is done. The Caspian brown trout, Salmo trutta caspius is [9] a critically endangered anadromous species which the cryopreservation of semen is a strategy for [24] permanent access to spermatozoa in this species. On the other hand, the dilution of semen during this process is necessary for better efficiency of crystallization. But, during the dilution, the majority of sperm cells lose their motility with time due to simultaneous activation in diluent solution. Fish spermatozoa are immotile in seminal fluid because of chemical properties of it. Thus, formulation of diluent solution (extender solution) mimic to specific properties of seminal fluid is necessary. Thus, in this study the values of chemical parameters of semen (ionic composition of seminal fluid, the ph and osmolality of semen) and their relationships with sperm motility characteristics (percentage and duration of motility) were investigated in Caspian brown trout. MATERIAL AND METHODS The experiment was carried out at the Kalardasht Salmonids Reproduction Center (KSRC), Iran, during the spawning season of Caspian brown trout. Males were captured from the Sardabrood and Tonekabon Rivers during their up stream reproductive migration, and then transferred to the broodstock pond at the KSRC. The 12 tagged males (TL: 60.9 ± 5.3 cm, TW: ± gr) were selected with occurrence of first spermiation and stripped repeatedly every 124 days during the spawning season (altogether five times during the spawning season). Before stripping, the fish were anaesthetized in 100 ppm of MS222 (tricaine methane sulfonate) then semen samples were collected by massage from the anterior portion of the testis towards the genital papilla. Care was taken to avoid contamination of the semen with water, mucus, blood cells, faeces or urine. Immediately after semen collection, the osmolality and ph of semen were measured with an osmometer (M elting Point Osmometer Nr , Roebling Company, Berlin, Germany) and a semi-microelectrode (SM102 ph Meter) respectively. A two-step dilution was used for motility activation according to method that suggested [4] by Billard and Cosson for salmonid fish. Firstly, the semen was prediluted to a physiological saline (composed of 80 mm NaCl, 40 mm KC1, l mm Corresponding Author: Saeed Hajirezaee, Department of Fisheries and Environmental Sciences, Faculty of Natural Resources, University of Tehran, PO Box Karaj, Iran. Shrezaee@yahoo.com 27

2 CaC1 2, and 20 mm Tris-HCl per one liter of distilled water (final ph= 9)) in a ratio of 1 /100 and secondly, the prediluted semen was subjected to a second dilution in a physiological serum at a ratio of 1/20 and immediately 1µl of solution was placed on the microscope stage and motility was analyzed by Semiquantitative method. In this regard, the motility was [23] recorded by a videocamera coupled with the optical lens of microscope. At the end, the video recordings were reviewed and the motility was presented as the percentage and duration of motility. Only forwardmoving sperm were judged motile, those simply vibrating or turning on their axes was considered [1] immotile. To analyse the ionic composition of seminal fluid, the semen was separated from the seminal fluid by centrifugation (Heraeus, Sepatech, Labofuge200, Germany, 5000 rpm for 10 min). Magnesium, chloride and calcium were measured with colorimetric method using an Auto-analyser Technican (RA 1000, Technicon-Swords, Dublin, Ireland). Potassium and Sodium were determined with a flamephotometer (CORNING 480, Corning, Medfield, MA, USA). The relationships between chemical properties of the seminal fluid and sperm motility characteristics (the percentage of motile spermatozoa and duration of motility) were tested using the bivariate correlation coefficients of Pearson. Then, the Linear and non-linear regression models were investigated using regression fits. The total duration of sperm motility and the percentage of motile spermatozoa were used as dependent and the parameters of seminal fluid as independent variables. RESULTS AND DISCUSSION The minimum, maximum and mean of the percentage (5s after activation) and duration of motility, ion composition, ph and the osmolality of the seminal fluid of Caspian brown trout have been shown in Table 1. From 57 semen samples, the percentage and the duration of spermatozoa motility in the ten samples were more than 70 % and 40 second respectively which the semen parameters of these ten samples have been shown in Table 2. Significant positive relationships were found for the percentage of motile - spermatozoa vs. Ca, Mg, K, osmolality, Cl, Na, and ph of semen (Table 3, p<0.01), also, the duration of motility vs. ph of semen (Figure 1). The differences in the concentrations of inorganic components of semen samples of fish could be related to the differences in secretary activity of the spermatic duct epithelium of individuals since the formation of the seminal fluid in fish (inorganic as well as organic compounds) is a secretion process of the spermatic [16,17,11,12] duct epithelium as these differences were observed between the semen samples of Caspian brown trout. In Caspian brown trout, significant positive relationships were observed between the duration of motlity and ph of semen, also between the percentage - of motile spermatozoa and Na, Cl, K, Ca, Mg, osmolality of semen. In agreement with our results, similar relationships were found in other fish. for example, the % motile spermatozoa vs. Na, K, ph, [14] osmolality in rainbow trout, % motile spermatozoa vs. ph in Chinook salmon, Oncorhynchus tshawytscha [8] -, % motile spermatozoa vs. Na, Mg Cl, % motile spermatozoa vs. Na, K, ph, osmolality in Alburnus [13] alburnus, duration of motility vs. ph in the rainbow [18,19] trout and chum salmon, Oncorhynchus keta, although the negative relationships were found for the percentage of motility vs. Ca, Mg, Na, K in [21] European eel. Fish seminal fluid has a unique composition regarding the presence of the organic and inorganic components which support the viability of spermatozoa [20,22,25,11,7,15]. In this regard, interactions of ions present in the seminal fluid with the sperm membrane do [7] influence the membrane potential and represent a mechanism of inhibition of spermatozoa in the seminal [3] fluid or sperm duct, allowing the maintenance of the potential of motility before release to the surrounding [2 0] medium. According to past data, it was revealed that the low K and low osmolality levels of surrounding medium are key factors for activation of salmonid and cyprinid fish spermatozoa at the time of [2,20] fertilization respectively. also, it is suggested that the increase of semen ph due to the secretion of bicarbonate ion into the seminal fluid may be responsible for acquisition of motility in the rainbow trout Oncorhynchus mykiss and chum salmon, [18,19,4] Oncorhynchus keta before sperm ejaculation i.e. during passage of spermatozoa from the testis to the spermatic duct. With regard to this point, the highly significant relationships between percentage of motile spermatozoa and K and also the duration of motility and ph suggest that these parameters may be the most important seminal fluid characteristics influencing on the potential of motility of Caspian brown trout spermatozoa before sperm ejaculation. Thus, the K and ph of semen could be two main indicators of semen quality of Caspian brown trout. Nevertheless, the positive relationship between the osmolality of semen and the percentage of motility shows that the motility of Caspian brown trout spermatozoa could be affected by osmolality of semen in semen fluid. 28

3 Fig. 1: Relationship between the ph and duration of spermatozoa motility in Caspian brown trout (n = 57 samples (.independent variable: ph, dependent variable: duration of motility. Table1: Minimum, maximum and the mean of seminal fluid parameters and motility characteristics of Caspian brown trout (n = 57semen samples, 3 males yielded no semen in fifth stripping). Seminal fluid parameters Minimum Maximum Mean SD Percentage of motile spermatozoa Duration of motility(s) Na (mm.l ) Cl (mm.l ) K (mm.l ) Ca (mm.l ) M g (mm.l ) Osmolality (mosmol.kg ) ph Table 2: The values of Ions of the seminal fluid, ph and osmolality of semen in the ten semen samples with more than 70 % and 40 second motility respectively. N=10 K Na Cl Mg Ca osmolality ph % motility Duration of motility N N N N N N N N N N N

4 Table3: Relationships between the chemical properties of seminal fluid and percentage of motile spermatozoa in Caspian brown trout (n= 57 samples). Seminal fluid parameters Bivariate coefficient Regression function R square P value Na (mm.l ) y = 0.58x Cl (mm.l ) y = 0.29x K (mm.l ) y = 1.07x Ca (mm.l ) y = x x M g (mm.l ) y = 17.4x Osmolality (mosmol.kg ) y = 0.26x ph y = x x Conclusion: In the present study, from 57 semen samples, the percentage and the duration of spermatozoa motility in the 10 samples were more than 70 % and 40 second respectively. Therefore, with regard to relationships between seminal fluid parameters and sperm motility characteristics of Caspian brown trout, the range of seminal fluid parameters in these samples (i.e. 10 samples with more than 70 % and 40 s motility) could be used to formulate the specific extender solution for dilution of semen of this species. The proposed solution could be: - Na ( mm.l ), Cl ( mm.l ), K ( mm.l ), Ca (1.3-2 mm.l ), Mg (1.1.6 mm.l ), Osmolality ( mosmol.kg ), ph ( ). ACKNOWLEDGEM ENTS We are grateful to the manager (Mr Rezvani) and staff of the Kalardasht Salmonids Reproduction Center (KSRC), Iran, for their help in providing Caspian brown trout semen. Thanks are due to Mr. H. Niksirat for helpful comments during the experiment. REFERENCES 1. Aas, G.H., T. Refstie and B. Gjerde, Evaluation of milt quality of Atlantic salmon. Aquaculture, 95: Bayness, S.M., A.P. Stoss and A.P. Dawson, Rainbow trout, salmo gairdnerii Richardson spermatozoa: Effects of cations and ph on motility. J Fish Biolog, 19: Boitano, S. and C.K. Omoto, Membrane hyperpolarization activates trout sperm without an increase in intracellular ph, Journal of Cell Science, 98: Billard, R. and M.P. Cosson, Some problems related to the assessment of sperm motility in freshwater fish. Journal of Experimental Zoology, 261: Billard, R., J. Cosson and L. Crime, Motility of fresh and aged halibut sperm. Aquat Living Resour, 6: Billard, R., J. Cosson, L.W. Crim and M. Suquet,1995. Sperm physiology and quality. In Brood Stock Management and Egg and Larval Quality, Eds., Bromage N.R., R.J. Roberts. Blackwell Science, Oxford, pp: Ciereszko, A., J. Glogowski and K. Dabrowski, Cryopreservation of Aquatic Species. In Biochemical characteristics of seminal plasma and spermatozoa of freshwater fishes, Eds., Tiersch TR, P.M. Mazik. World Aquaculture Society, Baton Rouge, LA, USA, pp: Ingermann, R.L., D.C. Bencic and J.G. Gloud, Low seminal plasma buffering capacity corresponds to high ph sensitivity of sperm motility in salmonids, Fish Physiology and Biochemistry, 24: Kiabi, B.H., A. Abdoli and M. Naderi, Status of fish fauna in the south Caspian basin of Iran, Zoology in the Middle East, 18: Krol, J., J. Glogowski, K. Demska-Zakes and P. Hliwa, Quality of semen and histological analysis of testes in Eurasian perch Perca luviatilis L. during a spawning period., Czech J. Anim. Sci, 51: Lahnsteiner, F., R.A. Patzner and T. Weismann,1993. The testicular main duct and the spermatic duct in some cyprinid fishes. II. Composition of seminal fluid, Journal of Fish Biology, 44: Lahnsteiner, F., R.A. Patzner and T. Weismann, The testicular main duct and the spermatic duct in cyprinid fishes I. Morphology, fine structure and histochemistry. J. Fish Biol., 44: Lahnsteiner, F., B. Berger, T. Weismann and R.A. Patzner, Motility of spermatozoa of Alburnus alburnus (Cyprinidae) and its relationship to sem inal plasm a compositio n and sp erm metabolism. Fish Physiology and Biochemistry, 15:

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