EFFECTS OF DILUTION RATIOS, DILUENTS AND OSMOLALITY ON SPERM MOTILITY OF COMMON CARP Cyprinus carpio

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1 THOÂNG BAÙO KHOA HOÏC EFFECTS OF DILUTION RATIOS, DILUENTS AND OSMOLALITY ON SPERM MOTILITY OF COMMON CARP Cyprinus carpio ABSTRACT ẢNH HƯỞNG CỦA TỈ LỆ PHA LOÃNG, MÔI TRƯỜNG VÀ THẨM THẤU LÊN HOẠT LỰC TINH TRÙNG CÁ CHÉP Cyprinus carpio Lê Minh Hoàng 1, Hoàng Hà Giang 2 Ngày nhận bài: 09/02/2015; Ngày phản biện thông qua: 08/6/2015; Ngày duyệt đăng: 15/12/2015 Sperm motility based assessment of the semen quality is a key indicator for fi sh artificial production and sperm preservation success. Sperm motility, in its turn, is affected by several elements that are required to be optimized for the best performance. The aim of this pilot study was to fi nd the suitable solution characterized by optimal dilution ratio, diluents, osmolality for the best sperm motility in Cyprinus carpio. Primarily, experiment with four test dilution ratio: 1:1; 1:25; 1:15; 1:100 (semen: distilled water) was conducted. After that, the optimal dilution ratio will be selected to apply in the following test on the effects of four diluents: 0.3% NaCl, 0.65% NaCl, 0.9% NaCl and distilled water. Finally, the effects of different osmolality: 100, 200, 300 and 400 mosm/kg; combining with the best dilution ratio and diluents from the previous experiments; were estimated. In conclusion, the best sperm motility was shown in solutions with dilution ratio of 1:25, sodium chloride 0.3% and osmolality of 100 mosm/kg. Furthermore, next experiments on effect of cations, ph and temperature were recommended to complete the optimization that can be applied for long term preservation of sperm in not only common carp but other teleost species. Keywords: Common carp, Cyprinus carpio, sperm motility, dilution ratio, diluents, osmolality TÓM TẮT Các phương pháp đánh giá chất lượng tinh dịch thông qua hoạt lực tinh trùng là chỉ số quan trọng cho sự thành công của sản xuất giống nhân tạo và bảo quản tinh. Hoạt lực tinh trùng, chính nó, lại chịu ảnh hưởng bởi một số yếu tố cần được tối ưu hóa để có kết quả tốt nhất. Mục đích của thí nghiệm thăm dò này là để tìm ra dung dịch phù hợp (về độ pha loãng, chất pha loãng, nồng độ thẩm thấu) cho hoạt lực tốt nhất ở tinh trùng cá chép Cyprinus carpio. Đầu tiên, thí nghiệm với bốn tỉ lệ pha loãng (tinh dịch: nước cất) 1:1; 1:25; 1:15; 1:100 đã được tiến hành. Sau đó, tỉ lệ pha loãng tối ưu sẽ được chọn để đưa vào thí nghiệm ảnh hưởng của bốn nồng độ pha loãng khác nhau gồm: 0.3% NaCl, 0.65% NaCl, 0.9% NaCl và nước cất. Cuối cùng, ảnh hưởng của bốn loại nồng độ thẩm thấu: 100, 200, 300 và 400 mosm/kg (tương đồng ở tỉ lệ pha loãng và nồng độ pha loãng tối ưu dựa trên kết quả của thí nghiệm trước đó) đã được đánh giá. Kết luận, tinh trùng hoạt lực tốt nhất được biểu hiện ở dung dịch với tỉ lệ pha loãng 1:25, NaCl 0.3% và nồng độ thẩm thấu là 100 mosm/kg. Ngoài ra, các thí nghiệm tiếp theo về ảnh hưởng của các cation, ph và nhiệt độ nên được tiến hành để hoàn chỉnh các điều kiện tối ưu áp dụng trong bảo quản tinh cá chép và những loài cá xương khác. Từ khóa: cá chép, Cyprinus carpio, hoạt lực tinh trùng, tỉ lệ pha loãng, chất pha loãng, nồng độ thẩm thấu 1 TS. Lê Minh Hoàng: Viện Nuôi trồng thủy sản Trường Đại học Nha Trang 2 ThS. Hoàng Hà Giang: Khoa Sau đại học Trường Đại học Nha Trang 34 TRƯỜNG ĐẠI HỌC NHA TRANG

2 I. INTRODUCTION Spermatozoa motility is a prerequisite parameter in determining fi sh semen quality and fertilizable capacity [10]. However, spermatozoa of both marine and freshwater fi sh species are immotile in the testis and semen plasma. Motility of spermatozoa only occurs after they are released into surrounding aqueous environment during natural reproduction or into a diluents in artifi cial reproduction [8]. In these environments, spermatozoa motility is infl uenced by several factors such as: temperature, dilution ratio, different diluents, ions concentration or osmolality [10]. Up to date, number of spermatozoa studies have been carried out on Persian surgeon - Acipenser persicus [2], European perch - Perca fl uviatilis [3] and yellow croaker - Larimichthys polyactis [10] but none of those on common carp (Cyprinus carpio). Common carp is an indigenous species, distributing in ponds, lakes and rivers from Northern to Southern of Viet Nam. Besides, it is also high value commercial on the local market. Nowadays, the indiscriminate exploitation of the wild with the introduction of new multi-line carp gradually results in losing pure native carp. This potential impact can be minimized by running programs to conserve the gene pool of the species population in any level in which gamete preservation can be an effective approach. Prior to this, the determination of the optimal environmental factors for sperm motility of common carp should be detected for enhancing the fertilize ability as well as effi cient preservation of sperm. Therefore, the aims of this study were to fi nd the best dilution ratio, diluents, osmotic pressure for carp sperm motility. II. MATERIALS AND METHODS All experiments were carried out at the Institute of Aquaculture in Nha Trang University from 20/2 to 2/6/2013. Semen of male brood fi sh in Earthern ponds in Ninh Phung Hatchery were stripped into 1.5 ml eppendorf, kept in ice bucket and immediately transferred to the laboratory for assessment. Original semen quality was primarily assessed by detecting spermatozoa motility (SM) immediately after semen were collected. The percentage of sperm exhibiting rapid, vigorous and forward movement was classifi ed under a microscope by diluting the sperm into distilled water at ratio 1:100 (mixing 1µl semen and 99 µl distilled water (DW)). Samples with the best performance of sperm then would be used for following experiments. * Effect of dilution ration on SM: To evaluate the effect of dilution ratio on motility, semen was diluted at ratio of 1:1, 1:25, 1:50 or 1:100 (semen: DW). SM was determined immediately after initiation of sperm activation until 100% of spermatozoa were immotile. * Effect of diluents on SM: To evaluate the effect of diluents on motility, semen was diluted in: NaCl 0.3%, NaCl 0.65%, NaCl 0.9% and DW at ratio 1:25 (semen: diluent). Then, SM was checked and analyzed. * Effect of osmolality on SM: To evaluate the effect of osmolality on motility, the semen was diluted at the ratio 1:25 in NaCl with concentrations of 100, 200, 300 or 400 mosm/kg. Then, SM was checked and analyzed. Data representing infl uence of dilution ratio, diluents and cations concentration on motility were analyzed by one-way ANOVA using SPSS Results are presented as mean±standard error. Differences with a probability value (P) of 0.05 (P<0.05) would be considered signifi cant. TRƯỜNG ĐẠI HỌC NHA TRANG 35

3 III. RESULTS AND DISCUSSION 1. Effect of dilution ratio on SM: Dilution is an important activator for the fish sperm motility and maintaining the fertilize ability of dilute semen in the water environment [10]. The effect of dilution on motility is shown in Figure 1. In this study, the best dilution ratio for carp SM was 1:25 with duration of sperm motility after activation was 59.2±25.4 seconds and the percentage of progressive sperm after 15 seconds was 92.5±27%, significantly different from observation in other dilution ratios (P<0.05). Durations of sperm motility after activated by diluting in 1:1; 1:50 and 1:100 were as long as around 40 seconds. In contrast, it was reported that, the best dilution ratio for Persian surgeon - Acipenser persicus and European perch Perca fluviatilis was 1:50 [2, 3], for yellow croaker Larimichthys polyactis was 1:100 [10]. This phenomenon was totally different from number of studies in which a linear correlation between fish sperm motility duration and dilution ratios had been well demonstrated (e.g. Prochilodus lineatus) [1, 12]. Based on these studies, it was quite clear that high dilution was required for synchronized activation of spermatozoa [2, 3, 12] while the low dilution could not activate the sperm motile completely [12]). Figure 1. Effect of dilution ratio on SM. The different alphabetic letters on each bar indicates significant difference between dilution ratios (P<0.05) 2. Effect of diluents on SM Diluent is also an important factor in stimulation SM. The different diluents mentioned in this study are characterized by the content and proportion of solubles which can activate the transportation of subtances in both sides of sperm membrane, hence activate the sperm motility. As can be seen in Figure 2, among four diluents, NaCl 0.3% shown the best performance in sperm of common carp with the duration of sperm motility was 1567 seconds and there were still nearly 94% progressive sperms during the fi rst 15 seconds. In addition, after 240 seconds, approximately 30 percent sperms were still motile. In this diluent, sperms were not only enhanced but also prolonged the motility. This observation was also shared by several previous studies on Cottus gobio [9], and common carp - Cyprinus carpio [4]. Although, in those studies, the used diluents were the mix of different subtances but the concentration of NaCl itself were similar to the present study. In conclusion, in diluent, NaCl play the most important role in sperm motility. 36 TRƯỜNG ĐẠI HỌC NHA TRANG

4 Figure 2. Effect of diluents on sperm motility of common carp The different alphabetic letters on each bar indicate significant differences between diluents (P<0.05) However, the NaCl concentration was increased, the poorer results were shown. For instance, there was only 70% sperm motility in diluent NaCl 0.65% after the fi rst 15 seconds. Then, the percentage of motile sperm was only half of those in NaCl 0.3%, and fi nally closer to zero after 240 seconds. Moreover, diluent NaCl 0.9% seemed not to be able to activate the sperm motility since only a few sperm was motile in the whole time. In distilled water, the sperms were initially activated as well as in NaCl 0.3% during the fi rst 15 seconds but after that the sperm motility was not remained. 3. Effect of osmotic pressure on SM Environmental osmolality was demonstrated to be involved in activation of sperm motility (reviewed by [7]). Sperm of many freshwater fi sh can be initiated by the hypotonic solutions and in cases, by isotonic media (reviewed by [1] and [7]). This could be a consequence of ecological adaptation that fertilization environment of most freshwater fi sh were hypotonic to their seminal plasma osmolality. Effect of osmolality on sperm motility of common carp was demonstrated by Figure 3. Figure 3. Effect of osmotic pressure on SM. The different alphabetic letters on each bar indicates significant difference between osmotic pressure levels (P<0.05). The solution with osmolality of 100 mosm/kg shown the best activation in which the percentage of sperm motility were always on top during the fi rst 180 seconds. This was followed by 200 mosm/kg osmolality solution. The solution with high osmolality (300 and 400 mosm/kg) seemed to inactivate the sperm in the whole time of observation. However, in these two high osmolality solutions, the duration of sperm motility (1200 TRƯỜNG ĐẠI HỌC NHA TRANG 37

5 and 800 seconds, respectively) were much signifi cant higher than in two remain solutions. This phenomenone could be explained by the osmoregulatory capacity of the sperm. Freshwater fi sh sperm is able to osmoregulate in isotonic or hypotonic environment but in hyperosmotic one. Hence, common carp sperms were better activated in two hypoosmotic solutions (bellow its seminal plasma osmolality of 286 mosm/kg [11]). However, there was an energy cost for osmoregulation, so that, eventually, in these solution, the sperm motility would more sharply decrease while in higher osmolality solutions, sperms were able to reserve their energy for longer motility [1]. Interestingly, in the osmolality of 200 mosm/kg which is more closer to the cellular osmolality of common carp sperm, sperms did not as active as in 100 mosm/kg but the duration of motility was much lower than the former ones. In comparision, the optimal osmolality for sperms of other species were much higher, such as, 365 mosm/kg in gilthead-sparus auratus, 300 mosm/kg in turbot - Solea senegalensis, mosm/ kg in cod - Gadus morhua [6]. The capacity of SM has demonstrated that it is depend on osmolality through the experiments using sugars or other compounds no containing ions [5]. IV. CONCLUSION AND RECOMENDATION In conclusion, the optimal dilution ratio, diluent and osmolality for activating sperm motility in common carp in this study were 1:25, NaCl 0.3% and 100 mosm/kg, respectively. We recommend that, further studies on effects of cations, ph, and temperature should be done to completely develop the optimization applied for long term preservation of sperm in not only common carp but other teleost species. REFERENCES 1. Alavi, S.M.H. and Cosson, J. (2006), Sperm motility in fishes. (II) Effects of ions and osmolality: A review, Cell Biology International. 30, pp Alavi, S.M.H., Cosson, J., Karami, M., Amiri, M.B. and Akhoundzadeh, M.A. (2004), Spermatozoa motility in the Persian sturgeon, Acipenser persicus: effects of ph, dilution rate, ions and osmolality., Reproduction. 128, pp Alavi, S.M.H., Rodina, M., Policar, T., Kozak, P., Psenicka, M. and Linhart, O. (2007), Semen of Perca fl uviatilis L.: Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility, Theriogenology. 68, pp Bastami, K.D., Imanpour, M.R. and Hoseinifar, S.H. (2010), Sperm of feral carp Cyprinus carpio: optimization of activation solution, Aquaculture International. 18, pp Cabrita, E., Robles, V. and Herráez, P. (2009), Sperm quality assessment, in Cabrit, E., Robles, V., and Herráe, P., Editors, Methods in Reproductive Aquaculture Marine and Freshwater Species, CRC Press Taylor & Francis Group, pp Cosson, J., Groison, L.A., Suquet, M., Fauvel, C., Dreanno, C. and Billard, R. (2008), Marine fish spermatozoa: racing ephemeral swimmers, Reproduction September , pp Dzyuba, V. and Cosson, J. (2014), Motility of fish spermatozoa: from external signaling to flagella response, Reprod Biol. 14, pp Islam, S.M. and Akhter, T. (2011), Tale of fish sperm and factors affecting Sperm Motility: A Review, Advances in Life Sciences. 1, pp Lahnsteiner, F., Berger, B., Weismann, T. and Patzner, A.R. (1997), Sperm structure and motility of the freshwater teleost Cottus gobio, Journal of Fish Biology. 50, pp Le, M.H., Lim, H.K., Min, B.H., Park, M.S., Son, M.H., Lee, J.U. and Chang, Y.J. (2011), Effects of varying dilutions, ph, temperature and cations on spermatozoa motility in fish Larimichthys polyactis, Environmental Biology. 32, pp Plouidy, M.G. and Billard, R. (1982), The chemical composition fluids of the gametes in the common carp, Cyprinus carpio, in Richter, C.J.J. and Goos, H.J.T., Editors, Reproductive biology of fishes, PUDOC, Wagenington. 12. Romagosa, E., Souza, B.E., Sanches, E.A., Baggio, D.M. and Bombardelli, R.A. (2010), Sperm motility of Prochilodus lineatus in relation to dilution rate and temperature of the activating medium, Journal of Applied Ichthyology. 26, pp TRƯỜNG ĐẠI HỌC NHA TRANG