Cryobiology 70 (2015) 184–189
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
New extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen q S. Judycka a, M. Szczepkowski b, A. Ciereszko a, G.J. Dietrich a,⇑ a b
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland Department of Sturgeon Fish Breeding Inland Fisheries Institute in Olsztyn, Pieczarki 50, 11-610 Pozezdrze, Poland
a r t i c l e
i n f o
Article history: Received 27 November 2014 Accepted 18 February 2015 Available online 25 February 2015 Keywords: Siberian sturgeon Sperm motility Cryopreservation Fertilization ability
a b s t r a c t The goal of this study was to develop a simple glucose–methanol extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen. Semen quality was assessed by determining post-thaw sperm motility and fertilizing ability at hatching stage. We tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in a methanol extender on post-thaw sperm motility. Sperm motility parameters and fertilizing ability of semen cryopreserved in 0.1 M glucose in 15% methanol (GM) were compared to previously described Tris–sucrose–KCl in 10% – methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 30 min equilibration in GM extender before cryopreservation and 30 min of post-thaw storage were determined. The beneficial effect of the glucose for semen cryopreservation was related to its concentration with a quite narrow optimum of 0.1 to 0.15 M. The fertilization rates of frozen/thawed sperm were similar for both (TSKM and GM) tested extenders. The sperm motility and fertilization rate were not affected either by 30 min equilibration in GM extender or by 30 min of post-thaw storage. Our work indicates that the use a simple extender consisting of 0.1 M glucose in 15% methanol can be an alternative cryopreservation method to those previously described for sturgeons. The use of an equilibration period and the possibility of post-thaw semen storage can improve organization of hatchery work and help with logistics of large-scale hatchery operations. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Sturgeon semen is characterized by several unique characteristics, including morphology, biochemistry and physiology [18]. Major morphologic features are the presence of acrosome [9,11], as well as an elongated nucleus and a midpiece with numerous mitochondria [8]. Other distinctive characteristics for sturgeon semen are low values of seminal plasma osmolality [14], protein concentration and sperm concentration [24]. Sturgeon spermatozoa are also characterized by prolonged duration of sperm motility and undergo acrosome reaction [20,26]. The uniqueness of sturgeon semen implicates a need for a specific approach in developing techniques regarding sperm cryopreservation. Several protocols of cryopreservation have been developed for sturgeon sperm [4]. Early studies on sturgeon sperm cryopreserva-
q Statement of funding: This work was supported by funds from the National Science Centre Granted on research project 2011/01/D/NZ9/03738 and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences. ⇑ Corresponding author. E-mail address: [email protected] (G.J. Dietrich).
http://dx.doi.org/10.1016/j.cryobiol.2015.02.005 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
tion were carried out in the 1960s [6] with limited success. Since the 90s, methods resulting in sufficient sperm post-thaw motility have been described [16,25,30]. The latter methods employed extenders consisting of potassium ions and sucrose buffered by Tris–HCl in methanol. Simple extenders containing glucose and methanol have been recently used for cryopreservation of salmonid fish sperm [10,13,22]. These results indicate that the buffering of extenders for cryopreservation to specific pH and the addition of potassium ions may not be necessary. However, it is unknown at present if the simple extenders can be useful for the effective cryopreservation of Siberian sturgeon (Acipenser baerii) semen. The specificity of sturgeon semen, especially regarding low osmolality of semen, requires an adjustment of cryopreservation procedures. Exceptionally, the selection of the optimal glucose concentration is very important in order to establish optimal osmolality of the extender. Individual samples of sturgeon milt are usually of high volume, which implicate the need for the cryopreservation of a large number of straws [24]. Thus, cryopreservation would benefit from a prolonged time from semen extension to freezing. It is also very important to test the post-thaw time of usefulness of cryopreserved semen for fertilization. Prolonged times of post-thaw storage would secure the fertilization of a large
S. Judycka et al. / Cryobiology 70 (2015) 184–189
batch of eggs. These improvements should deal with the logistics of large-scale hatchery operations [27]. The aim of this study was to develop a simple glucose– methanol extender for the cryopreservation of Siberian sturgeon semen. In the first experiment we aimed to optimize glucose concentration in the extender. In the second experiment sperm motility parameters and fertilizing ability of sperm cryopreserved in 0.1 M glucose in 15% methanol (GM) were compared to Tris– sucrose–KCl–methanol extender (TSKM). Additionally, we tested sperm motility and fertilization rate in relation to 30 min equilibration in GM extender and 30 min of post-thaw storage.
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Effects of extender composition on sperm motility and fertilization rate of fresh-diluted and frozen/thawed semen The experiment was conducted on 10.03.2014. Two extenders were prepared: GM (0.1 M glucose in 15% methanol) and TSKM (30 mM Tris, 23.4 mM sucrose, 0.25 mM KCl in 10% methanol, pH 8.0) [16]. The post-thaw sperm motility parameters and fertilizing ability of sperm cryopreserved in TSKM and GM extenders were tested on three individual samples of sperm (n = 3). Effect of 30 min equilibration of semen before freezing and 30 min time of post-thaw storage on sperm motility and fertilization rate of sperm cryopreserved in GM extender
Material and methods Source of gametes and measurements of sperm concentration The fish were maintained at the Dgal Aquaculture Facility of the Inland Fisheries Institute in Olsztyn, Poland. The experiments were conducted in March 2014 on six males (6–10 years old, body weight 4–12 kg) and three females (13–19 years old, body weight 20–25 kg). Before stimulation, the fish (both females and males) were transferred from ponds (water temperature 1.5 °C) to tanks. The water temperature in the tanks was gradually increased (1 °C/day) to 16 °C and then maintained at that temperature for 7 days. Next, the males were stimulated hormonally with an injection of 0.03 mg kg 1 using a synthetic analog LH-RHa (Sigma–Aldrich, USA). Milt was collected 24 h after hormonal stimulation using a syringe with an attached rigid tube inserted into the urogenital opening. Eggs were collected 24 h after stimulation with two doses of LH-RHa. The first dose contained 10% and the second 90% of hormone, and the total injection was 0.1 mg kg 1. Milt and eggs were collected on the 10th and 11th March, 2014 and were used for experiments within 1 h after collection. Approval was given by the Animal Experiments Committee in Olsztyn, Poland. Spermatozoa concentration was determined through counting in a Bürker chamber using 500 dilution of samples with 0.7% NaCl. Mean sperm concentration of fresh undiluted sperm was 0.86 109 ml 1. Cryopreservation of sperm Sperm samples from nine males (three sperm samples on 10.03.2014 and six sperm samples on 11.03.2014) were frozen individually. The cryopreservation was performed using a freezing unit for 20 straws which included an adjustable floating rack and Styrofoam box with an isolating Neopor block (MINITÜB GmbH, Tiefenbach, Germany). Sperm suspensions were sucked into 250 ll straws (IMV Technologies, L’Agile, France) and immediately placed on a floating set at 3 cm high above the surface of liquid nitrogen. After 5 min of cooling, the straws were transferred to liquid nitrogen and stored until sperm motility analysis and fertilization. Effect of glucose concentration in extender containing 15% methanol on fresh-diluted and frozen/thawed sperm motility parameters The experiment was conducted on 10.03.2014. The effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in 15% methanol (semen: extender 1:1) on the sperm motility of fresh-diluted and cryopreserved semen was tested on three individual samples of sperm (n = 3). The extender consisting of 0.1 M glucose and 15% methanol (GM) was arbitrarily chosen for the further experiments performed on 11.03.2014. The final concentration was 0.05 M and 7.5% for glucose and methanol, respectively.
The experiments were conducted on 11.03.2014. Semen was cryopreserved in GM extender both immediately after dilution and after 30 min of equilibration on ice at 4 °C. Sperm motility was measured for fresh-diluted and frozen/thawed sperm (n = 6). Fertilization Just before fertilization the straws were thawed in a water bath at a temperature of 40 °C for 5 s. Immediately after thawing, an adequate volume of frozen/thawed milt (100,000 spermatozoa/ egg) was added to about 100 eggs, diluted with an activation solution (described below) and left for 3 min. Next, the samples were washed with hatchery water and then with a tannic acid solution [12]. Egg samples (100 eggs per treatment) were stocked into separate baskets in an incubation apparatus. An undiluted fresh semen was used at the beginning and at the end of the fertilization trial to test the quality of the eggs. Fertilization rates were calculated as the percentage of hatched larvae after 7 days of incubation at 15 °C [12]. All trials were replicated twice. Sperm motility analysis The motility parameters of fresh sperm, fresh-diluted in extender, sperm equilibrated for 30 min, and frozen/thawed sperm were examined with computer-assisted sperm analysis (CASA) using the Hobson Sperm Tracker as described by Dietrich et al. [12]. Video recordings were made using a microscope with a 10 negative phase lense and a Sony CCD black-and-white video camera at 50 Hz frame rate. Semen was diluted a 1:50 with sperm-activating solution (10 mM Tris, 20 mM NaCl and 2 mM CaCl2, pH 8.5; [17]) supplemented with 0.5% bovine serum albumin to prevent the spermatozoa from sticking to the glass. After rapid mixing, 0.7 ll of this solution was immediately placed in a well of a 12-well multi-test glass slide (ICN Biomedicals Inc., Aurora, OH, USA) and cover-slipped. Sperm motility parameters were measured between 5 and 17 s post-activation. Video recordings were analyzed using the Hobson Sperm Tracker (Hobson Vision Ltd., Baslow, UK). The tracker simultaneously assessed 15 sperm motility parameters, but for simplicity only straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN = 100 VSL/ VCL), amplitude of lateral head displacement (ALH) and percentage of motile sperm (MOT) were chosen for further analysis. Analyses were run in duplicate for all of the experiments in the present study. Therefore, the value for each sample represents a mean from two separate measurements of motility parameters of 50 spermatozoa. Statistical analysis All the results are expressed as mean ± SD. All analyses were performed at a significance level of 0.05 using GraphPad Prism v. 6.0 (GraphPad Software Inc., San Diego, CA, USA). For statistical
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procedures data percentages were transformed by arcsine square root transformation. Data were subjected to repeated measures two-way analysis of variance (ANOVA) followed by Sidak’s post hoc test. The hatching success was compared using t-test.
Results Effect of glucose concentration in extender containing 15% methanol on fresh-diluted and frozen/thawed sperm motility parameters Glucose concentration in the extender did not have any effect on the percentage of sperm motility of fresh-diluted semen (about 80%) but significantly affected post-thaw sperm motility in a dosedependent way (range of 20–58%; Fig. 1). Application of glucose concentrations of 0 and 0.30 M resulted in percentages of post-thaw sperm motility that were about two times lower than concentrations of 0.10 and 0.15 M, while 0.20 M caused 20%
decrease of motile sperm. The sperm velocity parameters (VCL, VSL, VAP) were not affected either by concentration of glucose or by cryopreservation. Cryopreservation affected sperm trajectory parameters at concentrations of 0 and 0.30 M of glucose for ALH and LIN, respectively. Additionally, values of ALH for fresh semen decreased at 0.30 M glucose. The concentration of glucose at 0.10 M provided high and consistent results of post-thaw sperm motility and therefore the extender consisting of 0.1 M glucose in 15% methanol (GM) was selected to be used in subsequent experiments.
Effects of extender composition on sperm motility and fertilization rate of fresh-diluted and frozen/thawed semen Application of GM extender produced a 20% higher percentage of motile spermatozoa after freezing/thawing compared to TSKM extender (Fig. 2A). No differences in the percentage of motility of
Fig. 1. The effect of glucose concentrations in extender on fresh-diluted (s) and frozen/thawed (d) sperm motility parameters of Siberian sturgeon (n = 3). Different superscripts indicate statistical differences (P < 0.05) among fresh-diluted (a, b) and cryopreserved (x, y) semen. Asterisks indicate significant differences between the parameters for fresh-diluted and cryopreserved semen (P < 0.05).
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Fig. 2. The sperm motility (A) and fertilization rate (B) of fresh-diluted and frozen/thawed Siberian sturgeon sperm cryopreserved in GM and TSKM extenders (n = 3). Different superscripts (a, b, c) indicate statistical differences (P < 0.05) among fresh and cryopreserved semen.
fresh sperm diluted in both extenders were observed. The fertilization rate at hatching stage of cryopreserved semen was similar for both extenders and did not differ from fresh semen (Fig. 2B). Effect of 30 min equilibration of semen before freezing and 30 min time of post-thaw storage on sperm motility and fertilization rate of sperm cryopreserved in GM extender Cryopreservation caused a decrease (30–40%) in sperm motility (Fig. 3A). Equilibration of sperm in extender did not affect the sperm motility of either fresh-diluted or frozen/thawed semen. The percentage of hatched larvae for fresh semen did not differ from frozen/thawed sperm (Fig. 3B). The equilibration period did not have any influence on the fertilization rates. The percentage of post-thawed sperm motility (Fig. 4A) and fertilization rates (Fig. 4B) were not affected by 30 min of storage. Discussion In this study, we have developed an effective procedure for the cryopreservation of Siberian sturgeon sperm using a glucose– methanol extender. The selection of appropriate glucose concentration was a crucial step for developing a successful cryopreservation procedure. The fertilization rates of frozen/ thawed sperm were similar for both (TSKM and GM) tested extenders. A 30 min equilibration before cryopreservation did not have any influence on the quality of fresh-diluted and frozen/thawed
sperm. Additionally, the sperm motility and fertilization rate were not affected by 30 min of post-thaw storage. We demonstrated for the first time that Siberian sturgeon semen can be successfully cryopreserved using a simple glucose– methanol extender. The composition of extenders for the cryopreservation of sturgeons sperm usually consists of 23.4–30.0 mM sucrose and 0.25–1.0 mM KCl buffered by Tris–HCl supplemented with cryoprotectants (5–10% of methanol, ME2SO or DMA) [5,12,16]. Our work indicates that after the reduction of components in the extender to glucose and methanol this procedure can be an alternative cryopreservation method to those previously described for sturgeons. To our knowledge, the current study is a first report on the use of glucose for the cryopreservation of sturgeon sperm. Glucose is one of the sugars used as external cryoprotectants that stabilize the sperm cell membrane against cryoinjuries [7]. Recently cryopreservation procedures with the use of 0.18 M glucose in 9% methanol were successfully implemented for salmonid fish semen [13,22]. Our results demonstrate that glucose can be an efficient external cryoprotectant for sturgeon spermatozoa as well. An important finding of this study is the demonstration that the beneficial effect of the glucose is related to its concentration, with a quite narrow optimal concentration of 0.1–0.15 M. The low efficiency of glucose below 0.1 M can be explained by being insufficient concentration to demonstrate cryoprotective effect. On the other hand, the lower cryoprotective ability of higher concentrations of the glucose can be explained by an increase in osmolality,
Fig. 3. The effect of 30 min equilibration of semen in GM extender on sperm motility (A) and fertilization rate (B) of fresh-diluted and frozen/thawed Siberian sturgeon sperm (n = 6). Different superscripts (a, b) indicate statistical differences (P < 0.05) among fresh and cryopreserved semen.
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Fig. 4. The post-thaw sperm motility (A) and fertilization rate (B) of Siberian sturgeon sperm (n = 6). Different superscripts (a, b) indicate statistical differences (P < 0.05) among cryopreserved semen in different times after thawing.
which in sturgeon semen is much lower than in teleost fish [1]. Our results also demonstrated that contrary to the percentage of sperm motility, other motility parameters seem not to be related with glucose concentrations. Therefore it can be suggested that the glucose effects on spermatozoa during cryopreservation are mainly related to the activation of movement but not to parameters of motile spermatozoa such as velocity and trajectory parameters. Our simplified extender did not contain potassium ions, in contrast to commonly used extenders for sturgeons [2,15]. Similar to salmonid fish, the presence of potassium is required to maintain sturgeon spermatozoa in a quiescent state [21,29]. Decrease or removal of K+ ions at appropriate osmolality leads to sperm activation [2]. In our study, glucose–methanol extender did not activate sperm motility, which strongly suggests that the osmolality of the extender is mainly responsible for the maintenance of the spermatozoa in the quiescent stage and for this reason potassium ions are not required. We found that the equilibration of semen for 30 min in glucose– methanol extender did not affect sperm motility before cryopreservation. This is an important finding because sturgeon semen is of high volume and for this reason a prolonged time is needed to fill the straws. In the case of short equilibration only a limited number of straws can be filled at one time. An equilibration is rarely used for the cryopreservation of fish sperm due to a lack of improvement of post-thaw fertility [19] or even a decrease in the fertilizing ability of cryopreserved spermatozoa [3]. The latter suggested that interaction between extender constituents could be the reason for conflicting information about the usability of equilibration in the cryopreservation of fish spermatozoa. Our results showed that semen could be equilibrated at least 30 min before cryopreservation without loss of quality which allows quite a long handling time before freezing. Our results demonstrated that cryopreserved sperm can be stored without a decrease in quality for up to 30 min after thawing. It is recommended that cryopreserved semen of fish should be used for fertilization immediately after thawing [19,28]. To our knowledge, this is the first report regarding the verification of the effect of post-thaw storage on the sperm motility and fertilization ability of Siberian sturgeon semen. Recently Nynca et al. [23] in their study have demonstrated a similar phenomenon for salmonid fish. Since a similar extender was used it can be suggested that glucose–methanol extender is useful for the post-thaw storage of semen. The extended time of the usability of frozen/thawed sperm for fertilization can improve the organization of hatchery work because a high number of straws can be thawed and used for the fertilization of a large amount of eggs.
Summing up, our work indicates that Siberian sturgeon semen can be successfully cryopreserved with the application of a simple glucose–methanol extender. The concentration of glucose is important for cryopreservation efficiency. The use of an equilibration period and the possibility of post-thaw semen storage can improve the organization of hatchery work and help with the logistics of large-scale hatchery operations. Further studies should be focused on scaling up this cryopreservation technique for the facilitation of the artificial reproduction of Siberian sturgeon.
References [1] S.M.H. Alavi, J. Cosson, M. Karami, B.M. Amiri, M.A. Akhoundzadeh, Spermatozoa motility in the Persian sturgeon, Acipenser persicus: effects of pH, dilution rate, ions and osmolality, Reproduction 128 (2004) 819–828. [2] S.M.H. Alavi, M. Rodina, D. Gela, O. Linhart, Sperm biology and control of reproduction in sturgeon: (I) testicular development, sperm maturation and seminal plasma characteristics, Rev. Fish Biol. Fish. 22 (2012) 695–717. [3] I. Babiak, J. Glogowski, K. Goryczko, S. Dobosz, H. Kuzminski, J. Strzezek, W. Demianowicz, Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa, Theriogenology 56 (2001) 177–192. [4] R. Billard, J. Cosson, S.B. Noveiri, M. Pourkazemi, Cryopreservation and shortterm storage of sturgeon sperm, a review, Aquaculture 236 (2004) 1–9. [5] S. Boryshpolets, B. Dzyuba, M. Rodina, S.M.H. Alavi, D. Gela, O. Linhart, Cryopreservation of sterlet (Acipenser ruthenus) spermatozoa using different cryoprotectants, J. Appl. Ichthyol. 27 (2011) 1147–1149. [6] I.A. Burtsev, A.V. Serebryakova, First results of sturgeon sperm cryopreservation, in: E.V. Vladimirskaya (Ed.), Works of Young Scientists, VNIRO, Moscow, Russia, 1969, pp. 94–100 (in Russian). [7] E. Cabrita, L. Anel, M.P. Herraez, Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm, Theriogenology 56 (2001) 623–635. [8] G.N. Cherr, W.H. Clark, An acrosome reaction in sperm from the white sturgeon, acipenser-transmontanus, J. Exp. Zool. 232 (1984) 129–139. [9] A. Ciereszko, K. Dabrowski, S.D. Mims, J. Glogowski, Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum), Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 125 (2000) 197–203. [10] A. Ciereszko, G.J. Dietrich, J. Nynca, S. Dobosz, T. Zalewski, Cryopreservation of rainbow trout semen using a glucose-methanol extender, Aquaculture 420 (2014) 275–281. [11] T.A. Dettlaff, A.S. Ginzburg, O.I. Schmalhausen, Sturgeon Fishes: Developmental Biology and Aquaculture, Springer-Verlag, Berlin; New York, 1993. 67–71. [12] G.J. Dietrich, A. Ciereszko, R.K. Kowalski, A. Rzemieniecki, E. Bogdan, W. Demianowicz, M. Dietrich, R. Kujawa, J. Glogowski, Motility and fertilizing capacity of frozen/thawed sperm of Siberian sturgeon after a short-time exposure of fresh semen to mercury and cadmium, J. Appl. Ichthyol. 28 (2012) 973–977. [13] G.J. Dietrich, J. Nynca, S. Dobosz, T. Zalewski, A. Ciereszko, Application of glucose–methanol extender to cryopreservation of semen of sex-reversed females rainbow trout results in high post-thaw sperm motility and fertilizing ability, Aquaculture 434 (2014) 27–32. [14] B. Dzyuba, J. Cosson, S. Boryshpolets, O. Bondarenko, V. Dzyuba, G. Prokopchuk, I. Gazo, M. Rodina, O. Linhart, In vitro sperm maturation in sterlet, Acipenser ruthenus, Reprod. Biol. 14 (2014) 160–163.
S. Judycka et al. / Cryobiology 70 (2015) 184–189 [15] V. Dzyuba, J. Cosson, Motility of fish spermatozoa: from external signaling to flagella response, Reprod. Biol. 14 (2014) 165–175. [16] J. Glogowski, R. Kolman, M. Szczepkowski, A. Horvath, B. Urbanyi, P. Sieczynski, A. Rzemieniecki, J. Domagala, W. Demianowicz, R. Kowalski, A. Ciereszko, Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol, Aquaculture 211 (2002) 367–373. [17] H. Jahnichen, D. Warnecke, E. Trolsch, K. Kohlmann, H. Bergler, H.J. Pluta, Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm, J. Appl. Ichthyol. 15 (1999) 204–206. [18] B.G.M. Jamieson, Fish Evolution and Systematics: Evidence from Spermatozoa: with a Survey of Lophophorate, Echinoderm, and Protochordate Sperm and an Account of Gamete Cryopreservation/Barrie G.M. Jamieson with Contributions by L. K.-P. Leung, Cambridge University Press, Cambridge, New York, 1991. [19] F. Lahnsteiner, B. Berger, T. Weismann, R. Patzner, The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation, J. Appl. Ichthyol. 12 (1996) 99–106. [20] O. Linhart, S.D. Mims, W.L. Shelton, Motility of spermatozoa from shovelnose sturgeon and paddlefish, J. Fish Biol. 47 (1995) 902–909. [21] M. Morisawa, K. Suzuki, S. Morisawa, Effects of potassium and osmolality on spermatozoan motility of salmonid fishes, J. Exp. Biol. 107 (1983) 105–113. [22] J. Nynca, G.J. Dietrich, S. Dobosz, J. Grudniewska, A. Ciereszko, Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen, Aquaculture 433 (2014) 62–65. [23] J. Nynca, G.J. Dietrich, S. Dobosz, T. Zalewski, A. Ciereszko, Effect of post-thaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm, Theriogenology 83 (2015) 253–256.
189
[24] B. Piros, J. Glogowski, R. Kolman, A. Rzemieniecki, J. Domagala, A. Horvath, B. Urbanyi, A. Ciereszko, Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa, Fish Physiol. Biochem. 26 (2002) 289–295. [25] M. Psenicka, G.J. Dietrich, M. Wojtczak, J. Nynca, M. Rodina, O. Linhart, J. Cosson, A. Ciereszko, Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO, Cryobiology 56 (2008) 251–253. [26] M. Psenicka, M. Rodina, O. Linhart, Ultrastructural study on the fertilisation process in sturgeon (Acipenser), function of acrosome and prevention of polyspermy, Anim. Reprod. Sci. 117 (2010) 147–154. [27] A. Shaliutina, M. Hulak, I. Gazo, P. Linhartova, O. Linhart, Effect of short-term storage on quality parameters, DNA integrity, and oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon sperm, Anim. Reprod. Sci. 139 (2013) 127–135. [28] J. Stoss, W. Holtz, Cryopreservation of rainbow-trout (Salmo-gairdneri) sperm.1. Effect of thawing solution, sperm density and interval between thawing and insemination, Aquaculture 22 (1981) 97–104. [29] G.P. Toth, A. Ciereszko, S.A. Christ, K. Dabrowski, Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions, Aquaculture 154 (1997) 337–348. [30] L.I. Tsvetkova, J. Cosson, O. Linhart, R. Billard, Motility and fertilizing capacity of fresh and frozen–thawed spermatozoa in sturgeons Acipenser baeri and A. ruthenus, J. Appl. Ichthyol. 12 (1996) 107–112.
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
New extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen q S. Judycka a, M. Szczepkowski b, A. Ciereszko a, G.J. Dietrich a,⇑ a b
Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-747 Olsztyn, Poland Department of Sturgeon Fish Breeding Inland Fisheries Institute in Olsztyn, Pieczarki 50, 11-610 Pozezdrze, Poland
a r t i c l e
i n f o
Article history: Received 27 November 2014 Accepted 18 February 2015 Available online 25 February 2015 Keywords: Siberian sturgeon Sperm motility Cryopreservation Fertilization ability
a b s t r a c t The goal of this study was to develop a simple glucose–methanol extender for cryopreservation of Siberian sturgeon (Acipenser baerii) semen. Semen quality was assessed by determining post-thaw sperm motility and fertilizing ability at hatching stage. We tested the effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in a methanol extender on post-thaw sperm motility. Sperm motility parameters and fertilizing ability of semen cryopreserved in 0.1 M glucose in 15% methanol (GM) were compared to previously described Tris–sucrose–KCl in 10% – methanol extender (TSKM). Additionally, sperm motility and fertilizing ability in relation to 30 min equilibration in GM extender before cryopreservation and 30 min of post-thaw storage were determined. The beneficial effect of the glucose for semen cryopreservation was related to its concentration with a quite narrow optimum of 0.1 to 0.15 M. The fertilization rates of frozen/thawed sperm were similar for both (TSKM and GM) tested extenders. The sperm motility and fertilization rate were not affected either by 30 min equilibration in GM extender or by 30 min of post-thaw storage. Our work indicates that the use a simple extender consisting of 0.1 M glucose in 15% methanol can be an alternative cryopreservation method to those previously described for sturgeons. The use of an equilibration period and the possibility of post-thaw semen storage can improve organization of hatchery work and help with logistics of large-scale hatchery operations. Ó 2015 Elsevier Inc. All rights reserved.
Introduction Sturgeon semen is characterized by several unique characteristics, including morphology, biochemistry and physiology [18]. Major morphologic features are the presence of acrosome [9,11], as well as an elongated nucleus and a midpiece with numerous mitochondria [8]. Other distinctive characteristics for sturgeon semen are low values of seminal plasma osmolality [14], protein concentration and sperm concentration [24]. Sturgeon spermatozoa are also characterized by prolonged duration of sperm motility and undergo acrosome reaction [20,26]. The uniqueness of sturgeon semen implicates a need for a specific approach in developing techniques regarding sperm cryopreservation. Several protocols of cryopreservation have been developed for sturgeon sperm [4]. Early studies on sturgeon sperm cryopreserva-
q Statement of funding: This work was supported by funds from the National Science Centre Granted on research project 2011/01/D/NZ9/03738 and funds appropriated to the Institute of Animal Reproduction and Food Research, Polish Academy of Sciences. ⇑ Corresponding author. E-mail address: [email protected] (G.J. Dietrich).
http://dx.doi.org/10.1016/j.cryobiol.2015.02.005 0011-2240/Ó 2015 Elsevier Inc. All rights reserved.
tion were carried out in the 1960s [6] with limited success. Since the 90s, methods resulting in sufficient sperm post-thaw motility have been described [16,25,30]. The latter methods employed extenders consisting of potassium ions and sucrose buffered by Tris–HCl in methanol. Simple extenders containing glucose and methanol have been recently used for cryopreservation of salmonid fish sperm [10,13,22]. These results indicate that the buffering of extenders for cryopreservation to specific pH and the addition of potassium ions may not be necessary. However, it is unknown at present if the simple extenders can be useful for the effective cryopreservation of Siberian sturgeon (Acipenser baerii) semen. The specificity of sturgeon semen, especially regarding low osmolality of semen, requires an adjustment of cryopreservation procedures. Exceptionally, the selection of the optimal glucose concentration is very important in order to establish optimal osmolality of the extender. Individual samples of sturgeon milt are usually of high volume, which implicate the need for the cryopreservation of a large number of straws [24]. Thus, cryopreservation would benefit from a prolonged time from semen extension to freezing. It is also very important to test the post-thaw time of usefulness of cryopreserved semen for fertilization. Prolonged times of post-thaw storage would secure the fertilization of a large
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batch of eggs. These improvements should deal with the logistics of large-scale hatchery operations [27]. The aim of this study was to develop a simple glucose– methanol extender for the cryopreservation of Siberian sturgeon semen. In the first experiment we aimed to optimize glucose concentration in the extender. In the second experiment sperm motility parameters and fertilizing ability of sperm cryopreserved in 0.1 M glucose in 15% methanol (GM) were compared to Tris– sucrose–KCl–methanol extender (TSKM). Additionally, we tested sperm motility and fertilization rate in relation to 30 min equilibration in GM extender and 30 min of post-thaw storage.
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Effects of extender composition on sperm motility and fertilization rate of fresh-diluted and frozen/thawed semen The experiment was conducted on 10.03.2014. Two extenders were prepared: GM (0.1 M glucose in 15% methanol) and TSKM (30 mM Tris, 23.4 mM sucrose, 0.25 mM KCl in 10% methanol, pH 8.0) [16]. The post-thaw sperm motility parameters and fertilizing ability of sperm cryopreserved in TSKM and GM extenders were tested on three individual samples of sperm (n = 3). Effect of 30 min equilibration of semen before freezing and 30 min time of post-thaw storage on sperm motility and fertilization rate of sperm cryopreserved in GM extender
Material and methods Source of gametes and measurements of sperm concentration The fish were maintained at the Dgal Aquaculture Facility of the Inland Fisheries Institute in Olsztyn, Poland. The experiments were conducted in March 2014 on six males (6–10 years old, body weight 4–12 kg) and three females (13–19 years old, body weight 20–25 kg). Before stimulation, the fish (both females and males) were transferred from ponds (water temperature 1.5 °C) to tanks. The water temperature in the tanks was gradually increased (1 °C/day) to 16 °C and then maintained at that temperature for 7 days. Next, the males were stimulated hormonally with an injection of 0.03 mg kg 1 using a synthetic analog LH-RHa (Sigma–Aldrich, USA). Milt was collected 24 h after hormonal stimulation using a syringe with an attached rigid tube inserted into the urogenital opening. Eggs were collected 24 h after stimulation with two doses of LH-RHa. The first dose contained 10% and the second 90% of hormone, and the total injection was 0.1 mg kg 1. Milt and eggs were collected on the 10th and 11th March, 2014 and were used for experiments within 1 h after collection. Approval was given by the Animal Experiments Committee in Olsztyn, Poland. Spermatozoa concentration was determined through counting in a Bürker chamber using 500 dilution of samples with 0.7% NaCl. Mean sperm concentration of fresh undiluted sperm was 0.86 109 ml 1. Cryopreservation of sperm Sperm samples from nine males (three sperm samples on 10.03.2014 and six sperm samples on 11.03.2014) were frozen individually. The cryopreservation was performed using a freezing unit for 20 straws which included an adjustable floating rack and Styrofoam box with an isolating Neopor block (MINITÜB GmbH, Tiefenbach, Germany). Sperm suspensions were sucked into 250 ll straws (IMV Technologies, L’Agile, France) and immediately placed on a floating set at 3 cm high above the surface of liquid nitrogen. After 5 min of cooling, the straws were transferred to liquid nitrogen and stored until sperm motility analysis and fertilization. Effect of glucose concentration in extender containing 15% methanol on fresh-diluted and frozen/thawed sperm motility parameters The experiment was conducted on 10.03.2014. The effect of glucose concentration (0, 0.10, 0.15, 0.20 and 0.30 M) in 15% methanol (semen: extender 1:1) on the sperm motility of fresh-diluted and cryopreserved semen was tested on three individual samples of sperm (n = 3). The extender consisting of 0.1 M glucose and 15% methanol (GM) was arbitrarily chosen for the further experiments performed on 11.03.2014. The final concentration was 0.05 M and 7.5% for glucose and methanol, respectively.
The experiments were conducted on 11.03.2014. Semen was cryopreserved in GM extender both immediately after dilution and after 30 min of equilibration on ice at 4 °C. Sperm motility was measured for fresh-diluted and frozen/thawed sperm (n = 6). Fertilization Just before fertilization the straws were thawed in a water bath at a temperature of 40 °C for 5 s. Immediately after thawing, an adequate volume of frozen/thawed milt (100,000 spermatozoa/ egg) was added to about 100 eggs, diluted with an activation solution (described below) and left for 3 min. Next, the samples were washed with hatchery water and then with a tannic acid solution [12]. Egg samples (100 eggs per treatment) were stocked into separate baskets in an incubation apparatus. An undiluted fresh semen was used at the beginning and at the end of the fertilization trial to test the quality of the eggs. Fertilization rates were calculated as the percentage of hatched larvae after 7 days of incubation at 15 °C [12]. All trials were replicated twice. Sperm motility analysis The motility parameters of fresh sperm, fresh-diluted in extender, sperm equilibrated for 30 min, and frozen/thawed sperm were examined with computer-assisted sperm analysis (CASA) using the Hobson Sperm Tracker as described by Dietrich et al. [12]. Video recordings were made using a microscope with a 10 negative phase lense and a Sony CCD black-and-white video camera at 50 Hz frame rate. Semen was diluted a 1:50 with sperm-activating solution (10 mM Tris, 20 mM NaCl and 2 mM CaCl2, pH 8.5; [17]) supplemented with 0.5% bovine serum albumin to prevent the spermatozoa from sticking to the glass. After rapid mixing, 0.7 ll of this solution was immediately placed in a well of a 12-well multi-test glass slide (ICN Biomedicals Inc., Aurora, OH, USA) and cover-slipped. Sperm motility parameters were measured between 5 and 17 s post-activation. Video recordings were analyzed using the Hobson Sperm Tracker (Hobson Vision Ltd., Baslow, UK). The tracker simultaneously assessed 15 sperm motility parameters, but for simplicity only straight line velocity (VSL), curvilinear velocity (VCL), average path velocity (VAP), linearity (LIN = 100 VSL/ VCL), amplitude of lateral head displacement (ALH) and percentage of motile sperm (MOT) were chosen for further analysis. Analyses were run in duplicate for all of the experiments in the present study. Therefore, the value for each sample represents a mean from two separate measurements of motility parameters of 50 spermatozoa. Statistical analysis All the results are expressed as mean ± SD. All analyses were performed at a significance level of 0.05 using GraphPad Prism v. 6.0 (GraphPad Software Inc., San Diego, CA, USA). For statistical
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procedures data percentages were transformed by arcsine square root transformation. Data were subjected to repeated measures two-way analysis of variance (ANOVA) followed by Sidak’s post hoc test. The hatching success was compared using t-test.
Results Effect of glucose concentration in extender containing 15% methanol on fresh-diluted and frozen/thawed sperm motility parameters Glucose concentration in the extender did not have any effect on the percentage of sperm motility of fresh-diluted semen (about 80%) but significantly affected post-thaw sperm motility in a dosedependent way (range of 20–58%; Fig. 1). Application of glucose concentrations of 0 and 0.30 M resulted in percentages of post-thaw sperm motility that were about two times lower than concentrations of 0.10 and 0.15 M, while 0.20 M caused 20%
decrease of motile sperm. The sperm velocity parameters (VCL, VSL, VAP) were not affected either by concentration of glucose or by cryopreservation. Cryopreservation affected sperm trajectory parameters at concentrations of 0 and 0.30 M of glucose for ALH and LIN, respectively. Additionally, values of ALH for fresh semen decreased at 0.30 M glucose. The concentration of glucose at 0.10 M provided high and consistent results of post-thaw sperm motility and therefore the extender consisting of 0.1 M glucose in 15% methanol (GM) was selected to be used in subsequent experiments.
Effects of extender composition on sperm motility and fertilization rate of fresh-diluted and frozen/thawed semen Application of GM extender produced a 20% higher percentage of motile spermatozoa after freezing/thawing compared to TSKM extender (Fig. 2A). No differences in the percentage of motility of
Fig. 1. The effect of glucose concentrations in extender on fresh-diluted (s) and frozen/thawed (d) sperm motility parameters of Siberian sturgeon (n = 3). Different superscripts indicate statistical differences (P < 0.05) among fresh-diluted (a, b) and cryopreserved (x, y) semen. Asterisks indicate significant differences between the parameters for fresh-diluted and cryopreserved semen (P < 0.05).
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Fig. 2. The sperm motility (A) and fertilization rate (B) of fresh-diluted and frozen/thawed Siberian sturgeon sperm cryopreserved in GM and TSKM extenders (n = 3). Different superscripts (a, b, c) indicate statistical differences (P < 0.05) among fresh and cryopreserved semen.
fresh sperm diluted in both extenders were observed. The fertilization rate at hatching stage of cryopreserved semen was similar for both extenders and did not differ from fresh semen (Fig. 2B). Effect of 30 min equilibration of semen before freezing and 30 min time of post-thaw storage on sperm motility and fertilization rate of sperm cryopreserved in GM extender Cryopreservation caused a decrease (30–40%) in sperm motility (Fig. 3A). Equilibration of sperm in extender did not affect the sperm motility of either fresh-diluted or frozen/thawed semen. The percentage of hatched larvae for fresh semen did not differ from frozen/thawed sperm (Fig. 3B). The equilibration period did not have any influence on the fertilization rates. The percentage of post-thawed sperm motility (Fig. 4A) and fertilization rates (Fig. 4B) were not affected by 30 min of storage. Discussion In this study, we have developed an effective procedure for the cryopreservation of Siberian sturgeon sperm using a glucose– methanol extender. The selection of appropriate glucose concentration was a crucial step for developing a successful cryopreservation procedure. The fertilization rates of frozen/ thawed sperm were similar for both (TSKM and GM) tested extenders. A 30 min equilibration before cryopreservation did not have any influence on the quality of fresh-diluted and frozen/thawed
sperm. Additionally, the sperm motility and fertilization rate were not affected by 30 min of post-thaw storage. We demonstrated for the first time that Siberian sturgeon semen can be successfully cryopreserved using a simple glucose– methanol extender. The composition of extenders for the cryopreservation of sturgeons sperm usually consists of 23.4–30.0 mM sucrose and 0.25–1.0 mM KCl buffered by Tris–HCl supplemented with cryoprotectants (5–10% of methanol, ME2SO or DMA) [5,12,16]. Our work indicates that after the reduction of components in the extender to glucose and methanol this procedure can be an alternative cryopreservation method to those previously described for sturgeons. To our knowledge, the current study is a first report on the use of glucose for the cryopreservation of sturgeon sperm. Glucose is one of the sugars used as external cryoprotectants that stabilize the sperm cell membrane against cryoinjuries [7]. Recently cryopreservation procedures with the use of 0.18 M glucose in 9% methanol were successfully implemented for salmonid fish semen [13,22]. Our results demonstrate that glucose can be an efficient external cryoprotectant for sturgeon spermatozoa as well. An important finding of this study is the demonstration that the beneficial effect of the glucose is related to its concentration, with a quite narrow optimal concentration of 0.1–0.15 M. The low efficiency of glucose below 0.1 M can be explained by being insufficient concentration to demonstrate cryoprotective effect. On the other hand, the lower cryoprotective ability of higher concentrations of the glucose can be explained by an increase in osmolality,
Fig. 3. The effect of 30 min equilibration of semen in GM extender on sperm motility (A) and fertilization rate (B) of fresh-diluted and frozen/thawed Siberian sturgeon sperm (n = 6). Different superscripts (a, b) indicate statistical differences (P < 0.05) among fresh and cryopreserved semen.
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Fig. 4. The post-thaw sperm motility (A) and fertilization rate (B) of Siberian sturgeon sperm (n = 6). Different superscripts (a, b) indicate statistical differences (P < 0.05) among cryopreserved semen in different times after thawing.
which in sturgeon semen is much lower than in teleost fish [1]. Our results also demonstrated that contrary to the percentage of sperm motility, other motility parameters seem not to be related with glucose concentrations. Therefore it can be suggested that the glucose effects on spermatozoa during cryopreservation are mainly related to the activation of movement but not to parameters of motile spermatozoa such as velocity and trajectory parameters. Our simplified extender did not contain potassium ions, in contrast to commonly used extenders for sturgeons [2,15]. Similar to salmonid fish, the presence of potassium is required to maintain sturgeon spermatozoa in a quiescent state [21,29]. Decrease or removal of K+ ions at appropriate osmolality leads to sperm activation [2]. In our study, glucose–methanol extender did not activate sperm motility, which strongly suggests that the osmolality of the extender is mainly responsible for the maintenance of the spermatozoa in the quiescent stage and for this reason potassium ions are not required. We found that the equilibration of semen for 30 min in glucose– methanol extender did not affect sperm motility before cryopreservation. This is an important finding because sturgeon semen is of high volume and for this reason a prolonged time is needed to fill the straws. In the case of short equilibration only a limited number of straws can be filled at one time. An equilibration is rarely used for the cryopreservation of fish sperm due to a lack of improvement of post-thaw fertility [19] or even a decrease in the fertilizing ability of cryopreserved spermatozoa [3]. The latter suggested that interaction between extender constituents could be the reason for conflicting information about the usability of equilibration in the cryopreservation of fish spermatozoa. Our results showed that semen could be equilibrated at least 30 min before cryopreservation without loss of quality which allows quite a long handling time before freezing. Our results demonstrated that cryopreserved sperm can be stored without a decrease in quality for up to 30 min after thawing. It is recommended that cryopreserved semen of fish should be used for fertilization immediately after thawing [19,28]. To our knowledge, this is the first report regarding the verification of the effect of post-thaw storage on the sperm motility and fertilization ability of Siberian sturgeon semen. Recently Nynca et al. [23] in their study have demonstrated a similar phenomenon for salmonid fish. Since a similar extender was used it can be suggested that glucose–methanol extender is useful for the post-thaw storage of semen. The extended time of the usability of frozen/thawed sperm for fertilization can improve the organization of hatchery work because a high number of straws can be thawed and used for the fertilization of a large amount of eggs.
Summing up, our work indicates that Siberian sturgeon semen can be successfully cryopreserved with the application of a simple glucose–methanol extender. The concentration of glucose is important for cryopreservation efficiency. The use of an equilibration period and the possibility of post-thaw semen storage can improve the organization of hatchery work and help with the logistics of large-scale hatchery operations. Further studies should be focused on scaling up this cryopreservation technique for the facilitation of the artificial reproduction of Siberian sturgeon.
References [1] S.M.H. Alavi, J. Cosson, M. Karami, B.M. Amiri, M.A. Akhoundzadeh, Spermatozoa motility in the Persian sturgeon, Acipenser persicus: effects of pH, dilution rate, ions and osmolality, Reproduction 128 (2004) 819–828. [2] S.M.H. Alavi, M. Rodina, D. Gela, O. Linhart, Sperm biology and control of reproduction in sturgeon: (I) testicular development, sperm maturation and seminal plasma characteristics, Rev. Fish Biol. Fish. 22 (2012) 695–717. [3] I. Babiak, J. Glogowski, K. Goryczko, S. Dobosz, H. Kuzminski, J. Strzezek, W. Demianowicz, Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa, Theriogenology 56 (2001) 177–192. [4] R. Billard, J. Cosson, S.B. Noveiri, M. Pourkazemi, Cryopreservation and shortterm storage of sturgeon sperm, a review, Aquaculture 236 (2004) 1–9. [5] S. Boryshpolets, B. Dzyuba, M. Rodina, S.M.H. Alavi, D. Gela, O. Linhart, Cryopreservation of sterlet (Acipenser ruthenus) spermatozoa using different cryoprotectants, J. Appl. Ichthyol. 27 (2011) 1147–1149. [6] I.A. Burtsev, A.V. Serebryakova, First results of sturgeon sperm cryopreservation, in: E.V. Vladimirskaya (Ed.), Works of Young Scientists, VNIRO, Moscow, Russia, 1969, pp. 94–100 (in Russian). [7] E. Cabrita, L. Anel, M.P. Herraez, Effect of external cryoprotectants as membrane stabilizers on cryopreserved rainbow trout sperm, Theriogenology 56 (2001) 623–635. [8] G.N. Cherr, W.H. Clark, An acrosome reaction in sperm from the white sturgeon, acipenser-transmontanus, J. Exp. Zool. 232 (1984) 129–139. [9] A. Ciereszko, K. Dabrowski, S.D. Mims, J. Glogowski, Characteristics of sperm acrosin-like activity of paddlefish (Polyodon spathula Walbaum), Comp. Biochem. Physiol. B: Biochem. Mol. Biol. 125 (2000) 197–203. [10] A. Ciereszko, G.J. Dietrich, J. Nynca, S. Dobosz, T. Zalewski, Cryopreservation of rainbow trout semen using a glucose-methanol extender, Aquaculture 420 (2014) 275–281. [11] T.A. Dettlaff, A.S. Ginzburg, O.I. Schmalhausen, Sturgeon Fishes: Developmental Biology and Aquaculture, Springer-Verlag, Berlin; New York, 1993. 67–71. [12] G.J. Dietrich, A. Ciereszko, R.K. Kowalski, A. Rzemieniecki, E. Bogdan, W. Demianowicz, M. Dietrich, R. Kujawa, J. Glogowski, Motility and fertilizing capacity of frozen/thawed sperm of Siberian sturgeon after a short-time exposure of fresh semen to mercury and cadmium, J. Appl. Ichthyol. 28 (2012) 973–977. [13] G.J. Dietrich, J. Nynca, S. Dobosz, T. Zalewski, A. Ciereszko, Application of glucose–methanol extender to cryopreservation of semen of sex-reversed females rainbow trout results in high post-thaw sperm motility and fertilizing ability, Aquaculture 434 (2014) 27–32. [14] B. Dzyuba, J. Cosson, S. Boryshpolets, O. Bondarenko, V. Dzyuba, G. Prokopchuk, I. Gazo, M. Rodina, O. Linhart, In vitro sperm maturation in sterlet, Acipenser ruthenus, Reprod. Biol. 14 (2014) 160–163.
S. Judycka et al. / Cryobiology 70 (2015) 184–189 [15] V. Dzyuba, J. Cosson, Motility of fish spermatozoa: from external signaling to flagella response, Reprod. Biol. 14 (2014) 165–175. [16] J. Glogowski, R. Kolman, M. Szczepkowski, A. Horvath, B. Urbanyi, P. Sieczynski, A. Rzemieniecki, J. Domagala, W. Demianowicz, R. Kowalski, A. Ciereszko, Fertilization rate of Siberian sturgeon (Acipenser baeri, Brandt) milt cryopreserved with methanol, Aquaculture 211 (2002) 367–373. [17] H. Jahnichen, D. Warnecke, E. Trolsch, K. Kohlmann, H. Bergler, H.J. Pluta, Motility and fertilizing capability of cryopreserved Acipenser ruthenus L. sperm, J. Appl. Ichthyol. 15 (1999) 204–206. [18] B.G.M. Jamieson, Fish Evolution and Systematics: Evidence from Spermatozoa: with a Survey of Lophophorate, Echinoderm, and Protochordate Sperm and an Account of Gamete Cryopreservation/Barrie G.M. Jamieson with Contributions by L. K.-P. Leung, Cambridge University Press, Cambridge, New York, 1991. [19] F. Lahnsteiner, B. Berger, T. Weismann, R. Patzner, The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation, J. Appl. Ichthyol. 12 (1996) 99–106. [20] O. Linhart, S.D. Mims, W.L. Shelton, Motility of spermatozoa from shovelnose sturgeon and paddlefish, J. Fish Biol. 47 (1995) 902–909. [21] M. Morisawa, K. Suzuki, S. Morisawa, Effects of potassium and osmolality on spermatozoan motility of salmonid fishes, J. Exp. Biol. 107 (1983) 105–113. [22] J. Nynca, G.J. Dietrich, S. Dobosz, J. Grudniewska, A. Ciereszko, Effect of cryopreservation on sperm motility parameters and fertilizing ability of brown trout semen, Aquaculture 433 (2014) 62–65. [23] J. Nynca, G.J. Dietrich, S. Dobosz, T. Zalewski, A. Ciereszko, Effect of post-thaw storage time and sperm-to-egg ratio on fertility of cryopreserved brook trout sperm, Theriogenology 83 (2015) 253–256.
189
[24] B. Piros, J. Glogowski, R. Kolman, A. Rzemieniecki, J. Domagala, A. Horvath, B. Urbanyi, A. Ciereszko, Biochemical characterization of Siberian sturgeon Acipenser baeri and sterlet Acipenser ruthenus milt plasma and spermatozoa, Fish Physiol. Biochem. 26 (2002) 289–295. [25] M. Psenicka, G.J. Dietrich, M. Wojtczak, J. Nynca, M. Rodina, O. Linhart, J. Cosson, A. Ciereszko, Acrosome staining and motility characteristics of sterlet spermatozoa after cryopreservation with use of methanol and DMSO, Cryobiology 56 (2008) 251–253. [26] M. Psenicka, M. Rodina, O. Linhart, Ultrastructural study on the fertilisation process in sturgeon (Acipenser), function of acrosome and prevention of polyspermy, Anim. Reprod. Sci. 117 (2010) 147–154. [27] A. Shaliutina, M. Hulak, I. Gazo, P. Linhartova, O. Linhart, Effect of short-term storage on quality parameters, DNA integrity, and oxidative stress in Russian (Acipenser gueldenstaedtii) and Siberian (Acipenser baerii) sturgeon sperm, Anim. Reprod. Sci. 139 (2013) 127–135. [28] J. Stoss, W. Holtz, Cryopreservation of rainbow-trout (Salmo-gairdneri) sperm.1. Effect of thawing solution, sperm density and interval between thawing and insemination, Aquaculture 22 (1981) 97–104. [29] G.P. Toth, A. Ciereszko, S.A. Christ, K. Dabrowski, Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions, Aquaculture 154 (1997) 337–348. [30] L.I. Tsvetkova, J. Cosson, O. Linhart, R. Billard, Motility and fertilizing capacity of fresh and frozen–thawed spermatozoa in sturgeons Acipenser baeri and A. ruthenus, J. Appl. Ichthyol. 12 (1996) 107–112.
