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The effect of cryopreservation extender on sperm motility and hatch success in northern pike (Esox lucius)
Aquaculture 514 (2020) 734482
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The effect of cryopreservation extender on sperm motility and hatch success in northern pike (Esox lucius)
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Beata Irena Cejkoa, Beata Sarosieka, Katarzyna Dryla, Sylwia Judyckaa, Bożena Szczepkowskab, ⁎ Mirosław Szczepkowskib, Radosław Kajetan Kowalskia, a b
Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland Department of Sturgeon Fish Breeding, The Stanisław Sakowicz Inland Fisheries Institute, Olsztyn, Poland
ARTICLE INFO
ABSTRACT
Keywords: Northern pike CASA Sperm Eggs Cryopreservation
Northern pike (Esox lucius) sperm equilibration and cryopreservation were conducted using a simple cryopreservation extender (CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris at pH 8.5 and supplemented with 10% of methanol. Sperm was diluted tenfold (1:9; sperm:CE), and a subset of the samples was equilibrated (over 15 min) and stored on crushed ice (+4 °C). The remainder of the samples were drawn into French straws of 250 μl capacity and subsequently frozen. Thawing of sperm was carried out in a hot water bath at 40 °C for five seconds. The control group was fresh sperm that was neither diluted in CE nor frozen. After equilibration and cryopreservation, sperm motility parameters were analyzed using a Computer-Assisted Sperm Analysis system. Fertilization using different sperm:egg ratios (i.e. 400, 200, 100, 50, 25 and 10 thousand sperm per egg) was performed to determine the fertilization capacity of sperm. Sperm motility parameters of northern pike remain at the same level in fresh, equilibrated and cryopreserved samples. On the other hand, average sperm longevity was longest in equilibrated (118.0 s.) and cryopreserved (124.5 s.) sperm in comparison to fresh sperm (84.7 s.). Sperm samples equilibrated in CE achieved higher (n = 4. P < .05) hatching rate at sperm:egg ratios equal to 25,000 and 10,000:1 (85.3 and 77.2%, respectively) as compared to fresh (67.7 and 61.2%, respectively) or cryopreserved (73.5, 65.5%, respectively) samples. This indicated that CE may be a promising option not only for cryopreservation but also for northern pike sperm storage and fertilization capacity improvement under controlled conditions.
1. Introduction The Northern pike (Esox Lucius) is one of the most important socioeconomic freshawater fish species inhabiting Eurasia and plays important role in controlling ichthyofaunal structure as the major predator of low-value fishes. On the other hand, northern pike fish is an important angling species (Salam and Davies, 1994). However, in recent years, the number of northern pike has significantly decreased due to environmental degradation and overfishing (Säisä et al., 2008). In the hatchery practice, the success of fish reproduction depends on a sufficient number of mature gametes that are collected under controlled conditions. However, there is potentially a high variability in eggs and sperm quantity and quality. For example, northern pikes caught from their natural environment do not always produce a sufficient amount of mature sperm (Babiak et al., 1997). A possible explanation of this phenomenon is that some males reach their maturity later, resulting in variation in individual sperm production (Cejko et al., 2018a). It is also true that wild broodstocks are caught from open waters and transported ⁎
to a hatchery, where they are kept in captivity until they reach sexual maturity. This manipulation may result in lowering the quality of gametes and its capacity for fertilization. Consequently, hatcheries require securing more males than minimally needed for reproduction, which in turn requires more space in the hatchery and greater costs associated with the anaesthetics or treatment agents. On the other hand, sperm cryopreservation may be used for storage of a sufficient amount of sperm with the best quality as long as necessary and its utilization to fertilize the egg at the most convenient time. Benefits related to cryopreservation of sperm concern not only the protection of collected sperm but also the possibility of using cryopreserved sperm in large-scale in fish production (Cabrita et al., 2010). Techniques to secure and maintain a high biological value of collected sperm might therefore help increase the efficiency of reproduction in the hatchery practice. Under controlled breeding, the main objective is to provide a sufficient number of mature gametes (sperm and eggs) to future fertilization and fry reproduction. Sperm dilution has been investigated as a means of fertilizing with increasing the number of eggs that can be
Corresponding author. E-mail address: [email protected] (R.K. Kowalski).
https://doi.org/10.1016/j.aquaculture.2019.734482 Received 21 March 2019; Received in revised form 27 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Motility (MOT), progressive motile sperm (PRG), curvilinear velocity (VCL), average path velocity (VAP), straight-linear velocity (VSL), movement linearity (LIN), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of fresh, equilibrated (not frozen) and cryopreserved (frozen/thawed) northern pike (Esox lucius) sperm. Data represent mean ± SD (P > .05).
fertilized from the volume of sperm. The dilution of sperm in an appropriate solution or artificial seminal plasma allows for the storage of sperm samples in reduced metabolism conditions, ensuring maintaining its fertilization capacity (Sarosiek et al., 2014, 2016; Cejko et al., 2018b). Sperm dilution in an appropriate extender is also required before cryopreservation in order to prepare sperm trials for storage and to protect cell membrane integrity against damage during freezing/ thawing processes (Xin et al., 2017). Sperm cryopreservation is applied in aquaculture for the conservation of genetic variability and biodiversity of breeders. Such a technique may also facilitate optimal quality sperm samples and transport to the hatcheries, as well as increase the availability of sperm, regardless of the female's maturity synchronization (Torres et al., 2016). In northern pikes, cryopreservation of stripped and testicular sperm using different cryopreservation extenders has been conducted and capacity of sperm for fertilization after freezing/thawing using different dilution ratios and sperm:egg ratios was analyzed (Babiak et al., 1995, 1997; Lahnsteiner, 2000; Lahnsteiner et al., 1998; Zhang et al., 2010). Babiak et al. (1997) reported significant differences in fertilization ability, ranging from 6.6 to 96.0% of cryopreserved sperm among individual males of northern pikes used as a donor of sperm. From a practical point of view, the use of sperm with uniformed high quality is beneficial, for the fertilization of a large number of eggs. This reduces the risk of the occurrences of a significant number of unfertilized eggs due to poor sperm quality. Due to the lack of a constant protocol of northern pike cryopreservation, leading to the lack of reproducible results, an improvements in this technique is required prior to practical aplication. Therefore, the aim of this study was to investigate whether the cryopreservation extender (CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol at pH 8.5 is effective for the northern pike sperm storage and cryopreservation. The specific characteristics of sperm motility parameters, sperm longevity and fertilization capacity in response to fresh, equilibration and cryopreserved sperm was also analyzed.
Woynarovich solution containing 68 mM NaCl and 50 mM urea at a pH of 7.7 and 180 mOsm kg−1 supplemented with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) used as an activation buffer (Cejko et al., 2016). For the activation of sperm motility, 1 μl of sperm was mixed with 100 μl of activation buffer and placed into the well of a multitest glass slide (12 wells of 30-μm depth in Teflon-coated slide glass, Tekdon, Myakka City, FL, USA) and covered with a coverslip. Next, recordings were made approximately 6 s after the activation of motility using a Basler 202 K (Ahrensburg, Germany) digital camcorder integrated with an Olympus BX51 (Tokyo, Japan) microscope (lens Plan FL N 20×/0.5 NH ph 1). The cryopreserved sperm motility was recorded 10 s post-activation due to the delay in motility propagation in these samples. Sperm longevity was measured from the beginning of the sperm contact with activation buffer to the time when < 1% of sperm remained motile. The recording speed was equal to 47 frames per second. The first 200 frames of each recording were then analyzed by the CASA system. For each sperm, sample motility was measured twice, and every CASA parameter was calculated based on these duplicate measurements. During analysis, samples of sperm were stored at a temperature of +4 °C using a thermomixer (ThermoMixer C, Eppendorf, Hamburg, Germany). Sperm was characterized by motility > 80% and was used in further experimentation. 2.3. Sperm equilibration and cryopreservation
2. Material and methods
The cryopreservation protocol was adopted from the earlier study (Judycka et al., 2016). After motility verification, sperm samples were diluted in cryopreservation extender (CE) tenfold (1:9; sperm:CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris at pH 8.5 and supplemented with 10% of methanol. Temperature of cryopreservation extender was +4 °C. One part of the samples (stored in 1.5 ml tubes) was equilibrated and stored on crushed ice (+4 °C), and the second part was drawn into French straws of 250 μl capacity and intended for freezing. The control group was fresh sperm, stored in 1.5 ml tubes at +4 °C, which was neither diluted in CE nor frozen. After dilution, sperm samples were left for 15 min, and cryopreservation was carried out on a floating frame 3 cm above a pool of liquid nitrogen for 4 min.
2.1. Origin of fish and spawner management
2.4. Sperm thawing and analysis
Wild northern pike males (n = 4, age > 3 years, body weight 0.5 kg) and females (n = 4, age > 3 years, body weight 2.4 kg) were caught using gillnets from Dgał Wielki Lake on March 20, 2017. After catching, the males and females were separated and placed in separate lake cages with a volume of about 1 m3 in the Department of the Sturgeon Research hatchery in Pieczarki (northeastern Poland). Males were recognized through releasing sperm by gentle abdominal pressure and females by the shape of the abdomen and urogenital region. The water temperature in the holding cages was maintained in the range of 7–10 °C.
Frozen sperm samples were thawed in a hot water bath at 40 °C for 5 s. Thawed sperm were analyzed using the CASA system. Sperm parameters were also calculated for fresh and equilibrated sperm. 2.5. Egg collection and fertilization using different sperm:egg ratio Eggs were collected from 4 mature females using the air stripping method described by Cejko et al. (2016). For fertilization, eggs which showed best morphometric characteristics (similar size and color) were chosen from each female. Each of the sperm experimental variant was used for the fertilization of two batches of eggs. Approximately 50 eggs per batch were fertilized with 400, 200, 100, 50, 25 and 10 thousand fresh, equilibrated or cryopreserved sperm samples per egg. Before fertilization, the sperm concentration (×109 ml−1) was determined using the cytometric method (Bürker chamber). After a 1:2000 dilution of sperm samples with 0.7% NaCl (Sigma-Aldrich, St. Louis, MO, USA), the concentrations were 13.9 × 109, 11.7 × 109, 14.2 × 109 and 23.6 × 109 ml−1 for the first, second, third and fourth sperm samples, respectively. The sperm:egg ratio was achieved by simple dilution of sperm with the appropriate amount of immobilizing solution, containing 130 mM NaCl, 25 mM NaHCO3, 40 mM KCl, 20 mM Tris, 2 mM CaCl2 and 1.5 mm MgCl2 × 6H2O (Kobayashi et al., 2004) which was
2.2. Sperm collection and analysis Before sperm collection, the fish (4 individuals) were anesthetized using 2-phenoxyethanol (Sigma-Aldrich, St. Louis, MO, USA) at 0.5 ml l−1. Next, sperm were collected using gentle abdominal pressure and was subjected to analysis using the Computer-Assisted Sperm Analysis (CASA) system (CRISMAS, ImageHouse, Birmingham, UK). Sperm motility (MOT, %), progressive motile sperm (PRG, %), curvilinear velocity (VCL, μm s−1), straight-linear velocity (VSL, μm s−1), average path velocity (VAP, μm s−1), movement linearity (LIN, %), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz) and sperm duration (seconds) were determined. For CASA analysis, sperm samples were activated using a 3
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hatching rate among the different sperm:egg ratio groups in equilibrated sperm. The hatching rate in these samples ranged from 77.2–87.9%. When cryopreserved sperm was used for fertilization, the significant lowest hatching rate (65.5%) was noted for 10 thousand sperm per egg used for fertilization. A significant differences were noted between equilibrated, fresh and cryopreserved sperm when 25 and 10 thousand sperm were used per egg (Fig. 3; P < .05).
used to prevent premature sperm motility activation during the fertilization procedure and provide equal (and high enough for mixing with eggs) volume of sperm suspension (10 μl) in all experimental variants. The egg to activation buffer ratio was equal to 1:2 (about 0.5 ml of egg and 1 ml of activation buffer). Ten minutes after fertilization, the egg samples were carefully washed twice in hatchery water. The samples were then submerged in hatchery water and incubated in an experimental incubation unit separately for each type of sperm and replication at a temperature of 12 °C. The fertilization trial was determined by calculating the percentage of hatching rate for fresh, equilibrated and cryopreserved sperm used for fertilization.
4. Discussion Results of the presented study indicated for the first time that application of simple CE is beneficial for northern pike sperm storage and cryopreservation. There were no differences in CASA parameters, which remained at a high level between fresh, equilibrated and cryopreserved sperm, although the sperm longevity was significantly higher after CE was used for sperm storage for 15 min in comparison to fresh sperm. It was also true that equilibration of sperm samples in CE shows higher hatching rate at sperm:egg ratios equal to 25,000 and 10,000:1 as compared to either fresh or cryopreserved samples. This indicated that CE may be a promising option not only for cryopreservation but also for northern pike sperm storage and fertilization capacity improvement under controlled conditions. In northern pike, sperm motility is usually activated using hatchery water or a Billard solution containing 1 mM CaCl2, 30 mM glycine, 154 mM NaCl, 20 mM Tris at pH 9.0 and osmolality at 350 mOsm kg−1. As shown by Billard (1978), after hatchery water was used in sperm activation, its motility ranged between 60 and 80%, and the sperm longevity did not exceed 80 s. Similarly, after the Billard solution was used as activation buffer, sperm motility did not exceed 80% although the sperm velocities remained at a reduced level at 140 and 65 μm s−1 for VCL and VSL, respectively (Dietrich et al., 2016). On the other hand, for the Woynarovich solution containing 68 mM NaCl and 50 mM urea at pH 7.7 and osmolality at 180 mOsm kg−1, the sperm motility of northern pike was above 95%, and its velocities were twice as high, reaching 280 and 200 μm s−1 for VCL and VSL, respectively (Cejko et al., 2018a). As shown in this study, poor sperm motility or sperm kinetic parameters may result from inadequate activation buffer used for northern pike sperm analysis under controlled conditions. It was also confirmed, in our earlier research, that northern pike reproduction under controlled conditions in a Woynarovich solution is recommended. After its application to egg fertilization, there was a statistically significantly higher fertilization rate when compared to the Billard solution (Cejko et al., 2016). In addition to sperm motility, sperm longevity is also an important factor for the capacity of sperm fertilization, since in environmental conditions where fertilization occurs, the micropyle remains open for only a few seconds to a minute. In this study, the sperm longevity for northern pike samples stored in CE increased after equilibration (125 s) and cryopreservation (130 s) in comparison to fresh sperm (80 s). This phenomenon was observed for the first time in northern pikes and likely results from the composition of CE used in the experiment. These results are contrary to pirapitinga-do-sul (Brycon opalinus) sperm stored in CE (1.05% NaCl, 6.55% glucose and 1.4 M methyl glycol), which resulted in a statistically significant reduction of sperm longevity in post-thaw (47 s) samples of sperm in comparison to equilibrated (117 s) and fresh sperm (120 s). The authors indicated that to confer protection during freezing and thawing, extenders should be at a higher osmolality, e.g. 365 mOsm kg−1. However, despite the use of such an extender, the sperm longevity after freezing was on a reduced level, which may result from the extender–cryoprotectant interaction. This interaction was not likely related to cellular permeation (Viveiros et al., 2012). Sperm cryopreservation success as measured by CASA data shows very high efficiency of the adopted methodology. After using simple CE containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol for sperm equilibration and cryopreservation, there were no significant differences in sperm motility and sperm kinetic parameters in comparison to fresh sperm. This is due to
2.6. Statistical analysis Analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA), and the results were presented as mean ± SD. Differences between fresh, equilibrated and cryopreserved sperm for CASA parameters and sperm longevity were compared using one-way ANOVA. Differences among different sperm:egg ratios used between fresh, equilibrated and cryopreserved sperm for hatching rate were analyzed using two-way ANOVA and Tukey's multiple comparison test. All data representing percentages were transformed using the arcsine transformation prior to statistical analysis. 3. Results 3.1. Effect of sperm equilibration and cryopreservation on motility and longevity Sperm motility parameters of northern pikes were at a similar level (no statistical differences) in fresh, equilibrated and cryopreserved samples (Fig. 1; P > .05). On the other hand, sperm longevity was statistically longer in equilibrated and cryopreserved samples comparison to fresh sperm (Fig. 2; P < .05). 3.2. Effect of sperm equilibration and cryopreservation on hatching rate for different sperm:egg ratios Reproduction success of northern pikes was determined by the sperm:egg ratio of different types of sperm used for fertilization. The significant lowest hatching rate was noted when 25 (61.2%) and 10 (67.7%) thousand fresh sperm per egg samples was used for fertilization (Fig. 3; P < .05). On the other hand, there were no differences in
Fig. 2. Sperm longevity of fresh, equilibrated (not frozen) and cryopreserved (frozen/thawed) northern pike (Esox lucius) sperm. Data represent mean ± SD. Different superscript letters (a, b) indicated statistical differences (P < .05). 4
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Fig. 3. Hatching rate (%, mean ± SD) of northern pike (Esox lucius) after fertilization with fresh, equilibrated (not frozen) and cryopreserved (frozen/ thawed) sperm at sperm:egg ratios: 400, 200, 100, 50, 25 and 10 thousand sperm per egg. Different letters a and b indicate statistical differences between sperm:egg ratio in a different type of sperm, whereas x, y and z indicate statistical differences between the sperm:egg ratio inside one type of sperm.
the fact that the relationship between frozen/thawed sperm and sperm mobility is related to the CE used for either sperm equilibration or cryopreservation. For example, Babiak et al. (1995) reported very low post-thaw motility (below 40%) of northern pike after using CE containing 0.29 mM CaCl2, 0.4 mM MgCl2x6 H2O, 0.5 mM Na2HPO4, 5.1 mM KCL, 11.7 mM NaCl, 0.2. mM citric acid and 20 mM glucose supplemented with 10% of egg yolk. The authors indicated that egg yolk addition to the CE does not exhibit protective properties of northern pike sperm motility. On the other hand, Dietrich et al. (2016) indicated that CE containing 180 mM glucose and 9% methanol was an appropriate extender for equilibration and cryopreservation of northern pike sperm over a 15 min period, since no difference in CASA parameters, i.e. VCL, VAP, and VSL, between fresh sperm was observed. Moreover, sperm velocities were lower (130, 100 and 50 μm s−1, respectively, for VCL, VAP, and VSL) than the values presented in our research (180, 100 and 160 μm s−1, respectively, for VCL, VAP, and VSL). On the other hand, in whitefish (Coregonus lavaretus) there was an increase of all CASA parameters (MOT, VCL, VAP, VSL, LIN, and ALH) after sperm equilibration and cryopreservation in the extender containing 180 mM glucose and 9% of methanol after 15 min when compared to fresh sperm. The authors of that study suggested a speciesspecific response to sperm motility parameters in equilibration and cryopreservation conditions (Dietrich et al., 2016). The relationship between post-thaw motility and fertilization capacity of northern pike depends on the CE used for sperm storage and the sperm:egg ratio used for fertilization (Babiak et al., 1995). Dietrich et al. (2016) reported no difference in fertilization rates between cryopreserved and fresh sperm of northern pike. Although after using 300 thousand:1 and 600 thousand:1 sperm:egg ratios, the authors indicated statistically significant decreases in the survival from the eyed-egg stage to the hatched stage from 80 to 95% in both cases. Such differences were not observed when a 150,000:1 sperm:egg ratio was used. In our study, the fertilization ratio of cryopreserved sperm shows the same effectiveness as the fresh samples. Moreover, equilibrated samples show higher fertilization efficiency at sperm:egg ratios of 25,000 and 10,000:1 when compared to either fresh or cryopreserved samples. This high fertilization efficiency of the equilibrated samples could be explained by the longest effective duration of sperm motility measured in these samples. It is plausible that prolonged sperm motility is a factor that can achieve a higher fertilization ratio when a low sperm to egg ratio is used. Our results are similar to the data presented by Erdahl and Graham (1987), where 26,000 motile sperm were successfully used for egg fertilization in northern pike. In such a case, in CE (0.2 g CaCl2·2H2O, 0.4 g MgCl2·6H2O,
0.5 g Na2HPO4, 5.1 g KCl, 11.7 g NaCl, 0.2 g citric acid, 20 g glucose, KOH 20 ml at 1.27 g/100 ml) sperm was diluted 256 times without the loss of fertility after 30 min of storage. To conclude, improvements in the cryopreservation method should focus on maximizing fertilization success with a limited sperm number. Our results corroborate data showing that the sperm:egg ratio of approximately 26,000:1 is optimal for pike egg fertilization (Erdahl and Graham, 1987). Moreover, we have shown for the first time that further decreasing the sperm:egg ratio is possible, if we use the sperm equilibrated in the simple CE containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol. Such a technique, which could be considered sperm boosting, is a potentially promising option for the hatcheries producing northern pike fry for propagation in the inland waters. Acknowledgments The presented study is supported by the project”Pneumatic method of fish stripping – possible application, influence on the gametes quality and quantity and the welfare of fish” (acronym: PNEUFISH) financed under Polish Operational Programme "Sustainable Development of the Fisheries Sector and Coastal Fishing Areas 2007-2013", financed by the European Fisheries Fund contract no. OR-61724-OR1400001/10 and funds appropriated to the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland. References Babiak, I., Glogowski, J., Luczynski, M.J., Kucharczyk, D., Luczynski, M., 1995. Cryopreservation of the milt of the northern pike. J. Fish Biol. 46, 819–828. Babiak, I., Glogowski, J., Luczynski, M.J., Luczynski, M., 1997. Effect of individual male variability on cryopreservation of northern pike, Esox lucius L., sper. Aquac. Res. 28, 191–197. Billard, R., 1978. Changes in structure and fertilizing ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture 14, 187–198. Cabrita, E., Sarasquete, C., Martínez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales, S., Herraez, M.P., 2010. Cryopreservation of fish sperm: Applications and perspectives. J. Appl. Ichthyol. 26, 623–635. Cejko, B.I., Sarosiek, B., Krejszeff, S., Judycka, S., Szczepkowski, M., Szczepkowska, B., Kowalski, R.K., 2016. Effects of different stripping methods of female and activation medium on fertilization success in northern pike (Esox lucius). Czech J. Anim. Sci. 10, 481–486. Cejko, B.I., Krejszeff, S., Żarski, D., Judycka, S., Targońska, K., Kucharczyk, D., 2018a. Effect of carp pituitary homogenate (CPH) and sGnRHa (Ovaprim) in the northern pike (Esox lucius) spermation stimulation and its effect on quantity and quality of sperm. Anim. Reprod. Sci. 193, 217–225. Cejko, B.I., Horváth, Á., Kollár, T., Kása, E., Lujić, J., Marinović, Z., Kowalski, R.K.,
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The effect of cryopreservation extender on sperm motility and hatch success in northern pike (Esox lucius)
T
Beata Irena Cejkoa, Beata Sarosieka, Katarzyna Dryla, Sylwia Judyckaa, Bożena Szczepkowskab, ⁎ Mirosław Szczepkowskib, Radosław Kajetan Kowalskia, a b
Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Olsztyn, Poland Department of Sturgeon Fish Breeding, The Stanisław Sakowicz Inland Fisheries Institute, Olsztyn, Poland
ARTICLE INFO
ABSTRACT
Keywords: Northern pike CASA Sperm Eggs Cryopreservation
Northern pike (Esox lucius) sperm equilibration and cryopreservation were conducted using a simple cryopreservation extender (CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris at pH 8.5 and supplemented with 10% of methanol. Sperm was diluted tenfold (1:9; sperm:CE), and a subset of the samples was equilibrated (over 15 min) and stored on crushed ice (+4 °C). The remainder of the samples were drawn into French straws of 250 μl capacity and subsequently frozen. Thawing of sperm was carried out in a hot water bath at 40 °C for five seconds. The control group was fresh sperm that was neither diluted in CE nor frozen. After equilibration and cryopreservation, sperm motility parameters were analyzed using a Computer-Assisted Sperm Analysis system. Fertilization using different sperm:egg ratios (i.e. 400, 200, 100, 50, 25 and 10 thousand sperm per egg) was performed to determine the fertilization capacity of sperm. Sperm motility parameters of northern pike remain at the same level in fresh, equilibrated and cryopreserved samples. On the other hand, average sperm longevity was longest in equilibrated (118.0 s.) and cryopreserved (124.5 s.) sperm in comparison to fresh sperm (84.7 s.). Sperm samples equilibrated in CE achieved higher (n = 4. P < .05) hatching rate at sperm:egg ratios equal to 25,000 and 10,000:1 (85.3 and 77.2%, respectively) as compared to fresh (67.7 and 61.2%, respectively) or cryopreserved (73.5, 65.5%, respectively) samples. This indicated that CE may be a promising option not only for cryopreservation but also for northern pike sperm storage and fertilization capacity improvement under controlled conditions.
1. Introduction The Northern pike (Esox Lucius) is one of the most important socioeconomic freshawater fish species inhabiting Eurasia and plays important role in controlling ichthyofaunal structure as the major predator of low-value fishes. On the other hand, northern pike fish is an important angling species (Salam and Davies, 1994). However, in recent years, the number of northern pike has significantly decreased due to environmental degradation and overfishing (Säisä et al., 2008). In the hatchery practice, the success of fish reproduction depends on a sufficient number of mature gametes that are collected under controlled conditions. However, there is potentially a high variability in eggs and sperm quantity and quality. For example, northern pikes caught from their natural environment do not always produce a sufficient amount of mature sperm (Babiak et al., 1997). A possible explanation of this phenomenon is that some males reach their maturity later, resulting in variation in individual sperm production (Cejko et al., 2018a). It is also true that wild broodstocks are caught from open waters and transported ⁎
to a hatchery, where they are kept in captivity until they reach sexual maturity. This manipulation may result in lowering the quality of gametes and its capacity for fertilization. Consequently, hatcheries require securing more males than minimally needed for reproduction, which in turn requires more space in the hatchery and greater costs associated with the anaesthetics or treatment agents. On the other hand, sperm cryopreservation may be used for storage of a sufficient amount of sperm with the best quality as long as necessary and its utilization to fertilize the egg at the most convenient time. Benefits related to cryopreservation of sperm concern not only the protection of collected sperm but also the possibility of using cryopreserved sperm in large-scale in fish production (Cabrita et al., 2010). Techniques to secure and maintain a high biological value of collected sperm might therefore help increase the efficiency of reproduction in the hatchery practice. Under controlled breeding, the main objective is to provide a sufficient number of mature gametes (sperm and eggs) to future fertilization and fry reproduction. Sperm dilution has been investigated as a means of fertilizing with increasing the number of eggs that can be
Corresponding author. E-mail address: [email protected] (R.K. Kowalski).
https://doi.org/10.1016/j.aquaculture.2019.734482 Received 21 March 2019; Received in revised form 27 August 2019; Accepted 4 September 2019 Available online 05 September 2019 0044-8486/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Motility (MOT), progressive motile sperm (PRG), curvilinear velocity (VCL), average path velocity (VAP), straight-linear velocity (VSL), movement linearity (LIN), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of fresh, equilibrated (not frozen) and cryopreserved (frozen/thawed) northern pike (Esox lucius) sperm. Data represent mean ± SD (P > .05).
fertilized from the volume of sperm. The dilution of sperm in an appropriate solution or artificial seminal plasma allows for the storage of sperm samples in reduced metabolism conditions, ensuring maintaining its fertilization capacity (Sarosiek et al., 2014, 2016; Cejko et al., 2018b). Sperm dilution in an appropriate extender is also required before cryopreservation in order to prepare sperm trials for storage and to protect cell membrane integrity against damage during freezing/ thawing processes (Xin et al., 2017). Sperm cryopreservation is applied in aquaculture for the conservation of genetic variability and biodiversity of breeders. Such a technique may also facilitate optimal quality sperm samples and transport to the hatcheries, as well as increase the availability of sperm, regardless of the female's maturity synchronization (Torres et al., 2016). In northern pikes, cryopreservation of stripped and testicular sperm using different cryopreservation extenders has been conducted and capacity of sperm for fertilization after freezing/thawing using different dilution ratios and sperm:egg ratios was analyzed (Babiak et al., 1995, 1997; Lahnsteiner, 2000; Lahnsteiner et al., 1998; Zhang et al., 2010). Babiak et al. (1997) reported significant differences in fertilization ability, ranging from 6.6 to 96.0% of cryopreserved sperm among individual males of northern pikes used as a donor of sperm. From a practical point of view, the use of sperm with uniformed high quality is beneficial, for the fertilization of a large number of eggs. This reduces the risk of the occurrences of a significant number of unfertilized eggs due to poor sperm quality. Due to the lack of a constant protocol of northern pike cryopreservation, leading to the lack of reproducible results, an improvements in this technique is required prior to practical aplication. Therefore, the aim of this study was to investigate whether the cryopreservation extender (CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol at pH 8.5 is effective for the northern pike sperm storage and cryopreservation. The specific characteristics of sperm motility parameters, sperm longevity and fertilization capacity in response to fresh, equilibration and cryopreserved sperm was also analyzed.
Woynarovich solution containing 68 mM NaCl and 50 mM urea at a pH of 7.7 and 180 mOsm kg−1 supplemented with 0.5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) used as an activation buffer (Cejko et al., 2016). For the activation of sperm motility, 1 μl of sperm was mixed with 100 μl of activation buffer and placed into the well of a multitest glass slide (12 wells of 30-μm depth in Teflon-coated slide glass, Tekdon, Myakka City, FL, USA) and covered with a coverslip. Next, recordings were made approximately 6 s after the activation of motility using a Basler 202 K (Ahrensburg, Germany) digital camcorder integrated with an Olympus BX51 (Tokyo, Japan) microscope (lens Plan FL N 20×/0.5 NH ph 1). The cryopreserved sperm motility was recorded 10 s post-activation due to the delay in motility propagation in these samples. Sperm longevity was measured from the beginning of the sperm contact with activation buffer to the time when < 1% of sperm remained motile. The recording speed was equal to 47 frames per second. The first 200 frames of each recording were then analyzed by the CASA system. For each sperm, sample motility was measured twice, and every CASA parameter was calculated based on these duplicate measurements. During analysis, samples of sperm were stored at a temperature of +4 °C using a thermomixer (ThermoMixer C, Eppendorf, Hamburg, Germany). Sperm was characterized by motility > 80% and was used in further experimentation. 2.3. Sperm equilibration and cryopreservation
2. Material and methods
The cryopreservation protocol was adopted from the earlier study (Judycka et al., 2016). After motility verification, sperm samples were diluted in cryopreservation extender (CE) tenfold (1:9; sperm:CE) containing 185 mM trehalose, 40 mM KCl, 20 mM Tris at pH 8.5 and supplemented with 10% of methanol. Temperature of cryopreservation extender was +4 °C. One part of the samples (stored in 1.5 ml tubes) was equilibrated and stored on crushed ice (+4 °C), and the second part was drawn into French straws of 250 μl capacity and intended for freezing. The control group was fresh sperm, stored in 1.5 ml tubes at +4 °C, which was neither diluted in CE nor frozen. After dilution, sperm samples were left for 15 min, and cryopreservation was carried out on a floating frame 3 cm above a pool of liquid nitrogen for 4 min.
2.1. Origin of fish and spawner management
2.4. Sperm thawing and analysis
Wild northern pike males (n = 4, age > 3 years, body weight 0.5 kg) and females (n = 4, age > 3 years, body weight 2.4 kg) were caught using gillnets from Dgał Wielki Lake on March 20, 2017. After catching, the males and females were separated and placed in separate lake cages with a volume of about 1 m3 in the Department of the Sturgeon Research hatchery in Pieczarki (northeastern Poland). Males were recognized through releasing sperm by gentle abdominal pressure and females by the shape of the abdomen and urogenital region. The water temperature in the holding cages was maintained in the range of 7–10 °C.
Frozen sperm samples were thawed in a hot water bath at 40 °C for 5 s. Thawed sperm were analyzed using the CASA system. Sperm parameters were also calculated for fresh and equilibrated sperm. 2.5. Egg collection and fertilization using different sperm:egg ratio Eggs were collected from 4 mature females using the air stripping method described by Cejko et al. (2016). For fertilization, eggs which showed best morphometric characteristics (similar size and color) were chosen from each female. Each of the sperm experimental variant was used for the fertilization of two batches of eggs. Approximately 50 eggs per batch were fertilized with 400, 200, 100, 50, 25 and 10 thousand fresh, equilibrated or cryopreserved sperm samples per egg. Before fertilization, the sperm concentration (×109 ml−1) was determined using the cytometric method (Bürker chamber). After a 1:2000 dilution of sperm samples with 0.7% NaCl (Sigma-Aldrich, St. Louis, MO, USA), the concentrations were 13.9 × 109, 11.7 × 109, 14.2 × 109 and 23.6 × 109 ml−1 for the first, second, third and fourth sperm samples, respectively. The sperm:egg ratio was achieved by simple dilution of sperm with the appropriate amount of immobilizing solution, containing 130 mM NaCl, 25 mM NaHCO3, 40 mM KCl, 20 mM Tris, 2 mM CaCl2 and 1.5 mm MgCl2 × 6H2O (Kobayashi et al., 2004) which was
2.2. Sperm collection and analysis Before sperm collection, the fish (4 individuals) were anesthetized using 2-phenoxyethanol (Sigma-Aldrich, St. Louis, MO, USA) at 0.5 ml l−1. Next, sperm were collected using gentle abdominal pressure and was subjected to analysis using the Computer-Assisted Sperm Analysis (CASA) system (CRISMAS, ImageHouse, Birmingham, UK). Sperm motility (MOT, %), progressive motile sperm (PRG, %), curvilinear velocity (VCL, μm s−1), straight-linear velocity (VSL, μm s−1), average path velocity (VAP, μm s−1), movement linearity (LIN, %), amplitude of lateral head displacement (ALH, μm), beat cross frequency (BCF, Hz) and sperm duration (seconds) were determined. For CASA analysis, sperm samples were activated using a 3
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hatching rate among the different sperm:egg ratio groups in equilibrated sperm. The hatching rate in these samples ranged from 77.2–87.9%. When cryopreserved sperm was used for fertilization, the significant lowest hatching rate (65.5%) was noted for 10 thousand sperm per egg used for fertilization. A significant differences were noted between equilibrated, fresh and cryopreserved sperm when 25 and 10 thousand sperm were used per egg (Fig. 3; P < .05).
used to prevent premature sperm motility activation during the fertilization procedure and provide equal (and high enough for mixing with eggs) volume of sperm suspension (10 μl) in all experimental variants. The egg to activation buffer ratio was equal to 1:2 (about 0.5 ml of egg and 1 ml of activation buffer). Ten minutes after fertilization, the egg samples were carefully washed twice in hatchery water. The samples were then submerged in hatchery water and incubated in an experimental incubation unit separately for each type of sperm and replication at a temperature of 12 °C. The fertilization trial was determined by calculating the percentage of hatching rate for fresh, equilibrated and cryopreserved sperm used for fertilization.
4. Discussion Results of the presented study indicated for the first time that application of simple CE is beneficial for northern pike sperm storage and cryopreservation. There were no differences in CASA parameters, which remained at a high level between fresh, equilibrated and cryopreserved sperm, although the sperm longevity was significantly higher after CE was used for sperm storage for 15 min in comparison to fresh sperm. It was also true that equilibration of sperm samples in CE shows higher hatching rate at sperm:egg ratios equal to 25,000 and 10,000:1 as compared to either fresh or cryopreserved samples. This indicated that CE may be a promising option not only for cryopreservation but also for northern pike sperm storage and fertilization capacity improvement under controlled conditions. In northern pike, sperm motility is usually activated using hatchery water or a Billard solution containing 1 mM CaCl2, 30 mM glycine, 154 mM NaCl, 20 mM Tris at pH 9.0 and osmolality at 350 mOsm kg−1. As shown by Billard (1978), after hatchery water was used in sperm activation, its motility ranged between 60 and 80%, and the sperm longevity did not exceed 80 s. Similarly, after the Billard solution was used as activation buffer, sperm motility did not exceed 80% although the sperm velocities remained at a reduced level at 140 and 65 μm s−1 for VCL and VSL, respectively (Dietrich et al., 2016). On the other hand, for the Woynarovich solution containing 68 mM NaCl and 50 mM urea at pH 7.7 and osmolality at 180 mOsm kg−1, the sperm motility of northern pike was above 95%, and its velocities were twice as high, reaching 280 and 200 μm s−1 for VCL and VSL, respectively (Cejko et al., 2018a). As shown in this study, poor sperm motility or sperm kinetic parameters may result from inadequate activation buffer used for northern pike sperm analysis under controlled conditions. It was also confirmed, in our earlier research, that northern pike reproduction under controlled conditions in a Woynarovich solution is recommended. After its application to egg fertilization, there was a statistically significantly higher fertilization rate when compared to the Billard solution (Cejko et al., 2016). In addition to sperm motility, sperm longevity is also an important factor for the capacity of sperm fertilization, since in environmental conditions where fertilization occurs, the micropyle remains open for only a few seconds to a minute. In this study, the sperm longevity for northern pike samples stored in CE increased after equilibration (125 s) and cryopreservation (130 s) in comparison to fresh sperm (80 s). This phenomenon was observed for the first time in northern pikes and likely results from the composition of CE used in the experiment. These results are contrary to pirapitinga-do-sul (Brycon opalinus) sperm stored in CE (1.05% NaCl, 6.55% glucose and 1.4 M methyl glycol), which resulted in a statistically significant reduction of sperm longevity in post-thaw (47 s) samples of sperm in comparison to equilibrated (117 s) and fresh sperm (120 s). The authors indicated that to confer protection during freezing and thawing, extenders should be at a higher osmolality, e.g. 365 mOsm kg−1. However, despite the use of such an extender, the sperm longevity after freezing was on a reduced level, which may result from the extender–cryoprotectant interaction. This interaction was not likely related to cellular permeation (Viveiros et al., 2012). Sperm cryopreservation success as measured by CASA data shows very high efficiency of the adopted methodology. After using simple CE containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol for sperm equilibration and cryopreservation, there were no significant differences in sperm motility and sperm kinetic parameters in comparison to fresh sperm. This is due to
2.6. Statistical analysis Analyses were performed using GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA), and the results were presented as mean ± SD. Differences between fresh, equilibrated and cryopreserved sperm for CASA parameters and sperm longevity were compared using one-way ANOVA. Differences among different sperm:egg ratios used between fresh, equilibrated and cryopreserved sperm for hatching rate were analyzed using two-way ANOVA and Tukey's multiple comparison test. All data representing percentages were transformed using the arcsine transformation prior to statistical analysis. 3. Results 3.1. Effect of sperm equilibration and cryopreservation on motility and longevity Sperm motility parameters of northern pikes were at a similar level (no statistical differences) in fresh, equilibrated and cryopreserved samples (Fig. 1; P > .05). On the other hand, sperm longevity was statistically longer in equilibrated and cryopreserved samples comparison to fresh sperm (Fig. 2; P < .05). 3.2. Effect of sperm equilibration and cryopreservation on hatching rate for different sperm:egg ratios Reproduction success of northern pikes was determined by the sperm:egg ratio of different types of sperm used for fertilization. The significant lowest hatching rate was noted when 25 (61.2%) and 10 (67.7%) thousand fresh sperm per egg samples was used for fertilization (Fig. 3; P < .05). On the other hand, there were no differences in
Fig. 2. Sperm longevity of fresh, equilibrated (not frozen) and cryopreserved (frozen/thawed) northern pike (Esox lucius) sperm. Data represent mean ± SD. Different superscript letters (a, b) indicated statistical differences (P < .05). 4
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Fig. 3. Hatching rate (%, mean ± SD) of northern pike (Esox lucius) after fertilization with fresh, equilibrated (not frozen) and cryopreserved (frozen/ thawed) sperm at sperm:egg ratios: 400, 200, 100, 50, 25 and 10 thousand sperm per egg. Different letters a and b indicate statistical differences between sperm:egg ratio in a different type of sperm, whereas x, y and z indicate statistical differences between the sperm:egg ratio inside one type of sperm.
the fact that the relationship between frozen/thawed sperm and sperm mobility is related to the CE used for either sperm equilibration or cryopreservation. For example, Babiak et al. (1995) reported very low post-thaw motility (below 40%) of northern pike after using CE containing 0.29 mM CaCl2, 0.4 mM MgCl2x6 H2O, 0.5 mM Na2HPO4, 5.1 mM KCL, 11.7 mM NaCl, 0.2. mM citric acid and 20 mM glucose supplemented with 10% of egg yolk. The authors indicated that egg yolk addition to the CE does not exhibit protective properties of northern pike sperm motility. On the other hand, Dietrich et al. (2016) indicated that CE containing 180 mM glucose and 9% methanol was an appropriate extender for equilibration and cryopreservation of northern pike sperm over a 15 min period, since no difference in CASA parameters, i.e. VCL, VAP, and VSL, between fresh sperm was observed. Moreover, sperm velocities were lower (130, 100 and 50 μm s−1, respectively, for VCL, VAP, and VSL) than the values presented in our research (180, 100 and 160 μm s−1, respectively, for VCL, VAP, and VSL). On the other hand, in whitefish (Coregonus lavaretus) there was an increase of all CASA parameters (MOT, VCL, VAP, VSL, LIN, and ALH) after sperm equilibration and cryopreservation in the extender containing 180 mM glucose and 9% of methanol after 15 min when compared to fresh sperm. The authors of that study suggested a speciesspecific response to sperm motility parameters in equilibration and cryopreservation conditions (Dietrich et al., 2016). The relationship between post-thaw motility and fertilization capacity of northern pike depends on the CE used for sperm storage and the sperm:egg ratio used for fertilization (Babiak et al., 1995). Dietrich et al. (2016) reported no difference in fertilization rates between cryopreserved and fresh sperm of northern pike. Although after using 300 thousand:1 and 600 thousand:1 sperm:egg ratios, the authors indicated statistically significant decreases in the survival from the eyed-egg stage to the hatched stage from 80 to 95% in both cases. Such differences were not observed when a 150,000:1 sperm:egg ratio was used. In our study, the fertilization ratio of cryopreserved sperm shows the same effectiveness as the fresh samples. Moreover, equilibrated samples show higher fertilization efficiency at sperm:egg ratios of 25,000 and 10,000:1 when compared to either fresh or cryopreserved samples. This high fertilization efficiency of the equilibrated samples could be explained by the longest effective duration of sperm motility measured in these samples. It is plausible that prolonged sperm motility is a factor that can achieve a higher fertilization ratio when a low sperm to egg ratio is used. Our results are similar to the data presented by Erdahl and Graham (1987), where 26,000 motile sperm were successfully used for egg fertilization in northern pike. In such a case, in CE (0.2 g CaCl2·2H2O, 0.4 g MgCl2·6H2O,
0.5 g Na2HPO4, 5.1 g KCl, 11.7 g NaCl, 0.2 g citric acid, 20 g glucose, KOH 20 ml at 1.27 g/100 ml) sperm was diluted 256 times without the loss of fertility after 30 min of storage. To conclude, improvements in the cryopreservation method should focus on maximizing fertilization success with a limited sperm number. Our results corroborate data showing that the sperm:egg ratio of approximately 26,000:1 is optimal for pike egg fertilization (Erdahl and Graham, 1987). Moreover, we have shown for the first time that further decreasing the sperm:egg ratio is possible, if we use the sperm equilibrated in the simple CE containing 185 mM trehalose, 40 mM KCl, 20 mM Tris and supplemented with 10% of methanol. Such a technique, which could be considered sperm boosting, is a potentially promising option for the hatcheries producing northern pike fry for propagation in the inland waters. Acknowledgments The presented study is supported by the project”Pneumatic method of fish stripping – possible application, influence on the gametes quality and quantity and the welfare of fish” (acronym: PNEUFISH) financed under Polish Operational Programme "Sustainable Development of the Fisheries Sector and Coastal Fishing Areas 2007-2013", financed by the European Fisheries Fund contract no. OR-61724-OR1400001/10 and funds appropriated to the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland. References Babiak, I., Glogowski, J., Luczynski, M.J., Kucharczyk, D., Luczynski, M., 1995. Cryopreservation of the milt of the northern pike. J. Fish Biol. 46, 819–828. Babiak, I., Glogowski, J., Luczynski, M.J., Luczynski, M., 1997. Effect of individual male variability on cryopreservation of northern pike, Esox lucius L., sper. Aquac. Res. 28, 191–197. Billard, R., 1978. Changes in structure and fertilizing ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture 14, 187–198. Cabrita, E., Sarasquete, C., Martínez-Páramo, S., Robles, V., Beirão, J., Pérez-Cerezales, S., Herraez, M.P., 2010. Cryopreservation of fish sperm: Applications and perspectives. J. Appl. Ichthyol. 26, 623–635. Cejko, B.I., Sarosiek, B., Krejszeff, S., Judycka, S., Szczepkowski, M., Szczepkowska, B., Kowalski, R.K., 2016. Effects of different stripping methods of female and activation medium on fertilization success in northern pike (Esox lucius). Czech J. Anim. Sci. 10, 481–486. Cejko, B.I., Krejszeff, S., Żarski, D., Judycka, S., Targońska, K., Kucharczyk, D., 2018a. Effect of carp pituitary homogenate (CPH) and sGnRHa (Ovaprim) in the northern pike (Esox lucius) spermation stimulation and its effect on quantity and quality of sperm. Anim. Reprod. Sci. 193, 217–225. Cejko, B.I., Horváth, Á., Kollár, T., Kása, E., Lujić, J., Marinović, Z., Kowalski, R.K.,
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