Effect of albumin and casein supplementation on the common carp Cyprinus carpio L. sperm motility parameters measured by CASA

Transcription

1 DOI /s Effect of albumin and casein supplementation on the common carp Cyprinus carpio L. sperm motility parameters measured by CASA Radosław Kajetan Kowalski Beata Irena Cejko Sławomir Krejszeff Beata Sarosiek Sylwia Judycka Katarzyna Targońska Dariusz Kucharczyk Jan Glogowski Received: 5 February 2013 / Accepted: 9 July 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract Adhesion of sperm to a glass surface can be manifested by the low percentage of motile spermatozoa and lower speed. This crucial factor could disprove the sperm quality prediction from computer-assisted sperm analysis (CASA) results. In our study, this was particularly observed when solution L was used. However, protein supplementation was effective in the reduction in sperm adhesion. We found that albumin increased the sperm motility at concentrations of 0.25 % in solution L and 0.5 % in solution P. Casein was effective in both solutions at 0.25 % concentration. However, in solution L, the motility was lower than that measured in solutions P and L with the supplementation of BSA at 0.25 and 0.5 %. BSA supplementation was especially beneficial in terms of higher speed values regardless of an activation solution. Casein used with solution P allows us to obtain similar results as in the samples activated with BSA supplementation. Casein supplementation to solution L did not increase CASA results to the level observed in the samples activated with BSA added to the solution L. Our data suggest that the selection of an activation solution is one of the most crucial steps in the experiments requiring CASA data. Depending on the composition, different protein supplements should be used to reduce adhesion of sperm to a glass surface. We recommend the use of BSA at the % concentration because at this level we obtained the highest CASA parameters in both activation media. Keywords Fish Sperm motility CASA Glass adhesion R. K. Kowalski (&) B. I. Cejko B. Sarosiek S. Judycka J. Glogowski Department of Gamete and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, ul. Bydgoska 7, Olsztyn, Poland r.kowalski@pan.olsztyn.pl S. Krejszeff K. Targońska D. Kucharczyk Department of Lake and River Fisheries, Faculty of Environmental Sciences and Fisheries, University of Warmia and Mazury, Olsztyn, Poland

2 Introduction The quality of gametes determines the fertilization success rate in all animals breed in captivity. The most common sperm quality indicators used in male fertility studies are computer-assisted sperm analysis (CASA) parameters of sperm movement (Kime et al. 1996). CASA parameter such as total speed of sperm was proven to be a fertility indicator for the Atlantic salmon (Gage et al. 2004). Therefore, sperm motility parameters are the most important indicator in helping us to predict the biological quality of the male gametes and consequently, their effectiveness in fertilization trials (Cosson 2004; Linhart et al. 2008). CASA systems developed for the sperm motility measurements have been around four decades. Since that, CASA was used to estimate spermatozoa motility by measurement of numerous motility parameters that have significant importance in egg fertilization (Gage et al. 2004; Król et al. 2009). Although characteristic to a given species, sperm motility parameters, indicated by computer-assisted sperm analysis, are subjected to changes induced by both environmental (Dietrich et al. 2010; Jarmołowicz et al. 2010; Dziewulska et al. 2011) and hormonal (Cejko et al. 2011) factors. The conditions of the spermatozoa activation solutions are important for egg fertilization and their improvements crucial for reproduction optimalization. Therefore, ph, osmotic pressure, and ionic composition should be considered when deciding on the optimal activation solutions (Perchec et al. 1995; Alavi and Cosson 2005; Bastami et al. 2010). Despite the composition of an activation solution, the main technical problem during CASA analysis is sperm stacking to a glass surface. To avoid this situation, albumin is often recommended to be added to an activation solution. However, there is a lack of information regarding a minimal effective concentration of albumin in fish sperm motility measurements. Moreover, this protein has antioxidant potential and ion chelating ability (Halliwell 1988), properties which might be unwanted in certain experiments. Therefore, we tested the effects of casein as a second-choice protein in the CASA analysis. Their biological activity is also related to the antioxidant action, however, in the distinct way from albumin (Almajano et al. 2007). Herein, we analyzed how different concentration of casein and albumin affected common carp Cyprinus carpio L. sperm motility parameters to find a minimal effective concentration of both proteins in CASA analysis. Materials and methods Milt from carp (n = 5) was collected during the spawning season in accordance with the procedure described by Cejko et al. (2011). Directly after milt collection, spermatozoa motility was analyzed by means of a computer system for CASA. Common carp milt was activated by 100 mm NaCl? 10 mm Tris; ph: 9.0; 199 mosm kg -1 (Lahnsteiner et al solution L) and 5 mm KCl? 45 mm NaCl? 30 mm Tris; ph: 8.0; 160 mosm kg -1 (Perchec et al solution P) with a different concentration of bovine serum albumin, i.e., BSA (0.25, 0.5, 1.0, 2.0 %) and casein (0.25, 0.5, 1.0, 2.0 %). In order to measure motility, a 1 ll milt sample was activated by the selected activation solution (25 ll), after which a 1 ll drop of the milt solution mixture was placed on a microscope slide. About 6 s after the activation of motility, recordings were made with use of a Basler 202 K digital camcorder integrated with an OLYMPUS BX51 microscope. The recording speed was equal to 47 frames s -1. The first 200 frames of each recording were then analyzed utilizing the CRISMAS program (ImageHouse Ltd.). We analyzed the following

3 CASA parameters: the percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, lm s -1 ), straight-line velocity (VSL, lm s -1 ), linearity (LIN, %), wobbling index (WOB, %), amplitude of lateral head displacement (ALH, lm), and beat cross frequency (BCF, Hz). Tukey s post hoc test was applied (oneway repeated measures Anova) to determine the significance of differences in the CASA parameters. Differences between CASA parameters measured at the same protein concentration in both solutions were tested with the use of paired t test. Results Percentage of motile spermatozoa increased when BSA supplementation was used in both activation solutions (Fig. 1). In solution L, motility increased from 27.8 to 80.9 % when only 0.25 % of BSA was added. In solution P, this increase was not that evident; however, without the presence of BSA, the motility percentage was about 72.7 %, whereas after BSA supplementation at concentration of 0.25 and 0.5 %, it increased to 83.5 and 90.5 %, respectively. The highest curvilinear sperm speed was recorded at the final concentration from 0.5 to 1 % of BSA. In the solution L, it was and lm s -1, and in the solution P, it was and lm s -1, respectively. The progressive motile spermatozoa numbers increased in solution L from 8.0 % (no BSA) to 24.8 and 31.9 % when 0.25 and 0.5 % of BSA was present in solution L. In solution P, initial PRG was higher and reached 28.1 %, while BSA supplementation at concentration of 0.25 and 0.5 % elevated this value up to 34 % and reached their maximum in the presence of 1 % of BSA where PRG was equal to 42.1 %. The ALH value also increased as soon as 0.25 % of BSA was used in both solutions. In solution L, the ALH value changed from 1.2 to 1.8 lm, and in solution P, it changed from 1.6 to 1.9 lm. Parameters such as LIN and WOB were affected at the lowest level by the addition of BSA. Casein supplementation caused the incensement of MOT parameters, bur for solution L this incensement was lower than for solution P (Fig. 2). In solution L, motility changed from 27.8 % when no addition was applied to 54.9 and 60.5 % when 0.25 and 1 % of casein was added. In solution P, these values reached 91.8 % when a small amount of casein at 0.25 % was added. The highest curvilinear speed of sperm was recorded at a concentration of casein from 0.5 to 2 % in solution P where it reached lm s -1 in samples measured in presence of 2 % of casein. In solution L, the highest curvilinear speed was recorded when 0.5 % of casein was added (202.2 lm s -1 ), and afterward, it slightly decreased in the presence of 2 % of casein (186.8). The PRG increased in solution P from 8.0 % in the control to 28.1 % when 0.25 % of casein supplementation was applied. In the solution L, PRG value was higher and reached 38.0 in the presence of 0.5 % casein and 43.4 % when 2 % of casein was used. The ALH value increased in both solutions after adding 0.25 % of casein. In solution L, ALH was changed from 1.2 to 1.6 lm, and in solution P, it changed from 1.6 to 1.9 lm. Parameters, BCF and LIN were less affected by the presence of casein. The parameter WOB remained constant for all treatments. Discussion Proteins supplementation was effective in the reduction in sperm adhesion. We found that albumin increased sperm motility at the concentration of 0.25 % in solution L and at the concentration of 0.5 % in solution P. Casein was effective in both solutions at 0.25 %

4 Fig. 1 Motility parameters measured by CASA with the supplementation of different amounts of BSA. Data represent as follows: the percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, lm s -1 ), straight-line velocity (VSL, lm s -1 ), linearity (LIN, %), wobbling index (WOB, %), amplitude of lateral head displacement (ALH, lm), and beat cross frequency (BCF, Hz). Asterisks indicate the level of statistical significance between values measured in solutions L and P at the same protein concentration (*P \ 0.05; **P \ 0.01; ***P \ 0.001) concentration. However, motility in solution L was lower than that measured in solution P and in solution L with BSA supplementation, which was especially beneficial in terms of higher speed values regardless of activation solution. Depending on the composition of

5 Fig. 2 Motility parameters measured by CASA with the supplementation of different amounts of casein. Data represent as follows: the percentage of motile sperm (MOT, %), progressively motile sperm (PRG, %), curvilinear velocity (VCL, lm s -1 ), straight-line velocity (VSL, lm s -1 ), linearity (LIN, %), wobbling index (WOB, %), amplitude of lateral head displacement (ALH, lm), and beat cross frequency (BCF, Hz). Asterisks indicate the level of statistical significance between values measured in solutions L and P at the same protein concentration (*P \ 0.05; **P \ 0.01; ***P \ 0.001) activation solutions, different protein supplements should be used for reducing the adhesion of sperm to a glass surface. Motility parameters measured by CASA can be affected by the activation of solution composition (Martinez-Pastor et al. 2008). This could be an effect of the solution as well as

6 sperm adhesion phenomenon. The adhesion of sperm to a glass surface can be manifested by the low percentage of motile spermatozoa and lower speed, which could result in inadequate data when CASA systems are applied for sperm motility measurements. When we compared motility percentage measured with the use of two solutions, we saw a clear difference in motility when no BSA supplementation is used. This difference disappeared when 0.25 or 0.5 % of BSA was applied, thus indicating that the stickiness of sperm to glass can be modulated by the ionic composition of the solutions. Furthermore, the motility parameters of sperm can possibly be affected by the specific action of the ionic composition of activation media (Alavi and Cosson 2006). The adhesion of sperm to glass seems to be also related to osmolality of the solution used. Lower osmolality in solution P caused a lower level of adhesion contrary to solution L with a higher osmolality level. However, as we have showed, it is possible to overcome problems with adhesion by simple BSA supplementation from 0.25 to 0.5 % into an activation solution. Casein used in combination with P solution allows us to obtain similar results as in the samples activated with BSA supplementation. Although proven to be useful (Chapeau and Gagnon 1987) in reducing sperm adhesion to glass, using BSA might also influence certain CASA analyses such as heavy metal ions effect on fish sperm motility (Abascal et al. 2007). Therefore, it is beneficial to have an alternative to BSA anti-adhesive reagent when CASA analysis-related sperm environmental factors are concerned. Casein might be such a reagent but with caution because such a supplementation to the solution L did not improve CASA results. We assume that the ability of casein to reduce adhesion depends on the ionic composition of the activation solutions. Our data suggest that selection of an activation solution is crucial in experiments requiring CASA data. The protein supplementation for reducing sperm adhesion depends on the composition of an activation solution. We recommend the use of BSA at the % concentration, because at this level we obtained the highest CASA parameters values in both activation media. Acknowledgments The presented study is supported by National Science Centre Grant N N References Abascal FJ, Cosson J, Fauvel C (2007) Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility. J Fish Biol 70: Alavi SMH, Cosson J (2005) Sperm motility in fishes. I. Effects of temperature and ph: a review. Cell Biol Int 29: Alavi SMH, Cosson J (2006) Sperm motility in fishes. II. Effects of ions and osmolality: a review. Cell Biol Int 30:1 14 Almajano MP, Delgado ME, Gordon MH (2007) Changes in the antioxidant properties of protein solutions in the presence of epigallocatechin gallate. Food Chem 101: Bastami KD, Imanpour MR, Hoseinifar SH (2010) Sperm of feral carp Cyprinus carpio: optimization of activation solution. Aquac Int 18: Cejko BI, Kowalski RK, Kucharczyk D, _Zarski D, Targońska K, Glogowski J (2011) Effect of time after hormonal stimulation on semen quality indicators of common carp, Cyprinus carpio L. (Actinopterygii: Cypriniformes: Cyprinidae). Acta Ichthyol Piscat 41:75 80 Chapeau C, Gagnon C (1987) Nitrocellulose and polyvinyl coatings prevent sperm adhesion to glass without affecting the motility of intact and demembranated human spermatozoa. J Androl 8:34 40 Cosson J (2004) The ionic and osmotic factors controlling motility of fish spermatozoa. Aquac Int 12:69 85 Dietrich GJ, Dietrich M, Kowalski RK, Dobosz S, Karol H, Demianowicz W, Glogowski J (2010) Exposure of rainbow trout milt to mercury and cadmium alters motility parameters and reproductive success. Aquat Toxicol 97:

7 Dziewulska K, Rzemieniecki A, Domagała J (2011) Sperm motility characteristics of wild Atlantic salmon (Salmo salar L.) and sea trout (Salmo trutta m. trutta L.) as a basic for milt selection. J Appl Ichthyol 27: Gage MJG, Macfarlance CP, Yeates S, Ward RG, Searle JB, Parker GA (2004) Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. Curr Biol 14:44 47 Halliwell B (1988) Albumin an important extracellular antioxidant? Biochem Pharmacol 37: Jarmołowicz S, Demska-Zakęś K, Kowalski RK, Cejko BI, Glogowski J, Zakęś Z (2010) Impact of dibutyl phthalate and benzyl butyl phthalate on motility parameters of sperm from the European pikeperch Sander lucioperca (L.). Arch Pol Fish 18: Kime DE, Ebrahimi M, Dysten K, Roelants I, Rurangwa E, Moore HDM, Ollevier F (1996) Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish; application to the effects of heavy metals. Aquat Toxicol 36: Król J, Kowalski R, Hliwa P, Dietrich G, Stabiński R, Ciereszko A (2009) The effects of commercial preparations containing two different GnRH analogues and dopamine antagonists on spermiation and sperm characteristics in the European smelt Osmerus eperlanus (L.). Aquaculture 286: Lahnsteiner F, Berger B, Weismann T, Patzner RA (1998) Determination of semen quality of the rainbow trout by sperm motility, seminal plasma parameters and spermatozoal metabolism. Aquaculture 163: Linhart O, Alavi SMH, Rodina M, Gela D, Cosson J (2008) Comparision of sperm velocity, motility and fertilizing ability between firstly and secondly activated spermatozoa of common carp (Cyprinus carpio). J Appl Ichthyol 24: Martinez-Pastor F, Cabrita E, Soares F, Anel L, Dinis MT (2008) Multivariate cluster analysis to study motility activation of Solea senegalensis spermatozoa: a model for marine teleosts. Reproduction 135: Perchec G, Julin C, Cosson J, André F, Billard R (1995) Relationship between sperm ATP content and motility of carp spermatozoa. J Cell Sci 108: Perchec G, Cosson MP, Cosson J, Jeulin C, Billard R (1996) Morfological and kinetic changes of carp (Cyprinus carpio) spermatozoa after initiation of motility in distilled water. Cell Motil Cytoskelet 35: