Keywords

1 Introduction

In commercial fish culture, effective broodfish management is a prerequisite for successful artificial breeding (Alavi et al. 2008a). The management of male broodfish is highly family-specific and depends upon gonad morphology, the neuro-endocrine and endocrine regulation of spermatogenesis and spermatozoa maturation, and the physiology and biochemistry of sperm (Alavi et al. 2008a, 2012; Ciereszko 2008). This chapter summarizes methods of sperm collection, handling of sperm in the hatchery, short-term storage and cryopreservation of sperm in Percidae, and reviews studies of physiology and biochemistry of seminal plasma, sperm motility, and energetics, as well as factors influencing sperm quality.

2 Semen Collection and Stripping

Prior to stripping, broodfish should be anesthetized for 3–10 min. Tricaine methanesulfonate (99–132 mg L−1), 2-phenoxyethanol solution (0.2–0.3 mL L−1), clove oil, Syzygium aromaticum (approximately 0.5 mL L−1), and propiscin (1.5–2 mL L−1 water) have been used for walleye, Eurasian perch, and pikeperch (Satterfield and Flickinger 1995b; Król et al. 2006; Bokor et al. 2007; Kazun and Siwicki 2001; Teletchea et al. 2009). The anesthetized fish should be rinsed in freshwater to remove residual anesthetic and wiped dry to avoid water contamination of semen during stripping. Semen is collected by applying gentle pressure on the abdomen, anterior to the anal pore. Initially released drops should generally be discarded, to avoid contamination by urine spontaneously released from the bladder (Alavi et al. 2007). The container of stripped semen should be placed on ice immediately. The broodfish may be left to recover in freshwater with a prophylactic treatment of 5 mg L−1 furacin and 0.5 % NaCl (Satterfield and Flickinger 1995b).

3 Spermatozoa Morphology and Fine Structure

Of 159 species in nine genera of Percidae (Lahnsteiner and Patzner 2008), spermatozoa morphology and ultrastructure have been studied only for Eurasian perch, Perca fluviatilis (Lahnsteiner et al. 1995; Hatef et al. 2011) and pikeperch, Sander lucioperca (Lahnsteiner and Mansour 2004; Křišťan et al. 2014) (Fig. 5.1). In both species, the spermatozoon is identified as an acrosomeless aquasperm and differentiated into head, small midpiece, and flagellum (Fig. 5.1Aa, Ba). The spermatozoon is asymmetrical, with the flagellum inserting mediolaterally into the head (Fig. 5.1Ab, Bb). The head of the spermatozoon is spherical and contains the genomic material. Head dimensions are greater in Eurasian perch (length: 1.9 ± 0.2 μm and width: 1.8 ± 0.1 μm) than in pikeperch (length 1.6–1.8 ± 0.2 μm, width 1.3–1.6 ± 0.1 μm). In Eurasian perch, a small cylindrical midpiece (diameter: 0.8 ± 0.1 μm) is located at the base of the head (Fig. 5.1Aa). In pikeperch, the midpiece is integrated into the head region or lateral to the head (Fig. 5.1Ba). The nucleus of pikeperch contains two invaginations, the satellite nuclear notches (Fig. 5.1Bb). Proximal and distal centrioles are present in the midpiece, each consisting of nine triplets of microtubules (Fig. 5.1Ab, Ac, Bb, and Bc). In pikeperch, the proximal centriole is inclined relative to the distal centriole at 110° compared to 90° in Eurasian perch. The distal centriole serves as a basal body for the formation of the flagellum. Lahnsteiner et al. (1995) have reported the existence of a mitochondrion surrounding the cytoplasmic channel and forming an incomplete ring in Eurasian perch, while Hatef et al. (2011) reported two mitochondria (Fig. 5.1Ab, Ac). In pikeperch, two to four mitochondria are irregularly arranged in the midpiece region (Fig. 5.1Bb, Bc). In both species, a cytoplasmic channel (diameter 0.4 ± 0.1 μm) is clearly visible around the midpiece and flagellum (Fig. 5.1Ab, Bb). The length of the flagellum in Eurasian perch (30–35 or 25.3–32.5 μm) and pikeperch (length: 33.2 ± 0.9 μm) is similar and consists of an axoneme surrounded by a plasma membrane (Lahnsteiner et al. 1995; Wirtz and Steinmann 2006; Křišťan et al. 2014) (Fig. 5.1Ae, Bb). The axoneme exhibits typical eukaryotic microtubule-based organization with a central pair of single microtubules surrounded by nine peripheral doublets of microtubules (Fig. 5.1Ad, Bd). Outer and inner dynein arms, radial spokes, and other ultrastructural features of the axoneme are observed in spermatozoa of both species. Well-developed lateral fin-like projections are observed in pikeperch spermatozoa (Fig. 5.1Ba, Bd). Although Lahnsteiner et al. (1995) reported similar structures in Eurasian perch spermatozoa, but micrographs obtained from cross sections of flagella of Eurasian perch spermatozoa do not reveal such structures (Hatef et al. 2011) (Fig. 5.1Aa, Ad).

Fig. 5.1
figure 1

Spermatozoon morphology and ultrastructure of (a) Eurasian perch, Perca fluviatilis and (b) pikeperch, Sander lucioperca (Hatef et al. 2011; Křišťan et al. 2012b). Figures Aa and Ba SEM of spermatozoon composed of head, midpiece and flagellum (JSM 6300 or JSM 7401-F, JEOL Ltd., Tokyo, Japan). Figures Ab–Ae and Bb–Bd are TEMs of longitudinal (b, e) or cross (c, d) sections of the mid-piece (b, c) and the flagellum (d, e) (JEOL 1010, JEOL Ltd., Tokyo, Japan). Ax axoneme, CC cytoplasmic channel, CM central microtubules, CPB central pair bridge, CPP central pair projections, DC distal centriole, F flagellum, Fi fin-like structure, IDA inner dynein arm, M mid-piece, Mt mitochondria, N nucleus, ODA outer dynein arm, PC proximal centriole, Pm plasma membrane, PM peripheral microtubules, RS radial spokes, sn satellite nuclear notches

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4 Semen Production

Mature Eurasian perch males of 89–117 g body mass produce 0.6–6.8 mL of semen (Alavi et al. 2007). Sperm dry weight (expressed as percent of wet weight of semen) in walleye (Sander vitreus) weighing 967–2747 g is reported to be between 5.3 % and 28.2 %, with a mean value of 20.8 % (Gregory 1970). The average volume of semen produced by walleyes in first, second, and third daily strippings is 3.1, 1.3, and 0.5 mL, respectively (Satterfield and Flickinger 1995b). Brown and Moore (1996) reported average values of 3.6 mL of semen per walleye male, but provided no information regarding broodfish body mass. Pikeperch weighing 778–1284 g produce 0.4–1.1 mL of semen (Korbuly et al. 2009). Most studies show high inter-individual variations in production of semen in Percidae.

Semen in Percidae is characterized by a high spermatozoa concentration, reaching 19–128 × 109 cells mL−1 in Eurasian perch, 40–75 × 109 cells mL−1 in yellow perch (Perca flavescens), and 21–69 × 109 cells mL−1 in walleye (Table 5.1). Reported spermatozoa concentration in other percid species is much lower, for example 4.3–20.6 × 109 cells mL−1 in pikeperch, 8.4 × 109 cells mL−1 in Volga pikeperch, and 13.2 × 109 cells mL−1 in hybrids of pikeperch and Volga pikeperch (Table 5.1). This may be due to the existence of a urinary bladder at the urogenital pore in pikeperch, which readily allows contamination of semen by urine during stripping (Křišťan et al. 2014).

Table 5.1 Ionic contents, pH and osmolality of the seminal plasma in Percidae
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High spermatozoa concentration in semen of percids implies a relatively low volume of seminal plasma, and spermatozoa volume can represent up to 95 % of the total volume of semen (Piironen and Hyvärinen 1983). Spermatocrit (the proportion of white packed material relative to the total volume of semen following centrifugation in a capillary tube) is reported to be 66–70 % in Eurasian perch (Hatef et al. 2010, 2011), and 36–94 % in walleye (Gregory 1970).

Wide variation in semen volume and spermatozoa concentration reported in different studies may be related to broodfish rearing conditions, methods of spawning induction, duration of broodfish participation in spawning, and duration and time of spermiation within the reproductive season (Alavi et al. 2008a; Ciereszko 2008).

5 Seminal Plasma Composition

Seminal plasma comprises both mineral and organic compounds produced by the testicular main duct and the sperm duct (Alavi et al. 2008a; Ciereszko 2008). It plays a key role in protecting and maintaining vital spermatozoa functions including viability, motility, and fertilizing ability for a prolonged period. Maintenance of a quiescent state, supply of adequate levels of nutrients, and protection against damage by microbes or xenobiotics are key elements for integrity of spermatozoa function (Wojtczak et al. 2005; Dietrich et al. 2011).

5.1 Mineral Composition of Seminal Plasma

Sodium ions predominate in the seminal plasma; their concentration is tenfold that of potassium ions (Table 5.1). Potassium ion concentration is five to tenfold that of calcium ions (Table 5.1). The osmolality of seminal plasma is similar among percids, with measured values of approximately 300 mOsmol kg−l in mid-spawning season (Table 5.1). It is assumed that seminal plasma osmolality is a major factor in maintaining spermatozoa in a quiescent state during storage in the reproductive system (see Sect. 5.6 in this chapter). The slight inter-species differences in seminal plasma osmolality among percids may be related to the amount of ions and inorganic compounds (Lahnsteiner et al. 1995; Ciereszko 2008).

5.2 Organic Composition of Seminal Plasma

5.2.1 Organic Substances with Low Molecular Weight

Cholesterol and monosaccharides (glucose, fructose, and galactose) are among numerous organic substances identified in the seminal plasma of Eurasian perch that are potentially important for steroid hormone biosynthesis and the energy of spermatozoa (Piironen and Hyvärinen 1983; Lahnsteiner et al. 1995; Henrotte et al. 2010). Piironen and Hyvärinen (1983) identified glycerol and citric acid in perch seminal plasma and linked the former with the lipolytic activity of testes and the latter with chelating of divalent ions, which have importance for keeping spermatozoa in a quiescent state (Ciereszko et al. 2000).

The amino acids alanine (2.1 mM), arginine (7.8 mM), asparagine (1.1 mM), cysteine (0.6 mM), glutamic acid (1.0 mM), isoleucine (1.1 mM), lysine (0.02 mM), methionine (1.6 mM), phenylalanine (0.3 mM), tryptophan (0.1 mM), and valine (0.5 mM) have recently been detected in the seminal plasma of Eurasian perch (Lahnsteiner 2010). Composition of free amino acids in the seminal plasma of Eurasian perch compared to other fish species highlights species-specific differences (Lahnsteiner 2009, 2010). It has been shown also that 48 h in vitro incubation of sperm in the presence of 2.5 mM of asparagine, lysine, methionine, or valine increases Eurasian perch spermatozoa motility and velocity (Lahnsteiner 2010). This is strong evidence of supporting roles of amino acids in spermatozoa viability. Further studies are required to investigate the physiological effects of specific amino acids on spermatozoa function (Lahnsteiner 2009, 2010).

Seminal plasma contains numerous low and high molecular weight antioxidative substances to protect spermatozoa. Uric acid has been identified in the seminal plasma of yellow perch (Ciereszko et al. 1999) and cysteine and glutathione in the semen of Eurasian perch and pikeperch (Stejskal et al. 2008). Lahnsteiner and Mansour (2010) identified ascorbic acid, glutathione, methionine, and uric acid and suggested a physiological role in the antioxidant system of Eurasian perch semen. Their studies show that uric acid is the most abundant, possibly being a major antioxidant in semen, as it improves sperm motility and membrane integrity and decreases sperm lipid peroxidation.

5.2.2 Proteins and Enzymes

Concentration of proteins in seminal plasma of Eurasian perch shows higher values than in other percids (Table 5.2). High protein concentration in seminal plasma correlates with high spermatozoa concentration in these species (Nynca et al. 2010).

Table 5.2 Protein concentration and anti-trypsin activity in seminal plasma of Percidae
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Similar to other teleosts (Dabrowski and Ciereszko 1994), seminal plasma of percids is characterized by a species-specific system of proteins with anti-trypsin activity. Anti-trypsin activity in Eurasian perch is one of the highest among percids (Table 5.2). Multiple forms of anti-trypsin activity are observed in the seminal plasma of percids; at least five compared to one to three in other species (Dabrowski and Ciereszko 1994; Ciereszko et al. 2000; Król et al. 2006; Nynca et al. 2010). The physiological importance of multiple forms of anti-trypsin activity is largely unknown, but it is species-specific and could potentially be used for the identification of hybrids of Percidae. The anti-trypsin activity profile in the seminal plasma shows similarities to that in the blood plasma. This suggests that inhibitors in seminal plasma can originate from blood. It is assumed that these inhibitors play key roles in protection of spermatozoa and reproductive tissue from proteolytic attack and/or in the regulation of spermatogenesis and spermatozoa motility (Ciereszko 2008). Potential targets for anti-trypsin activity in percid fish are proteolytic enzymes and proteolytic activity (Lahnsteiner et al. 1995; Kowalski et al. 2003, 2004; Król et al. 2011).

A significant correlation is shown between protein concentration and anti-trypsin activity in percids, including Eurasian perch and Volga pikeperch (Nynca et al. 2010). This association suggests that serine protease inhibitors are major proteins in the seminal plasma of percid fish and/or that their synthesis is regulated by the same mechanism as for other seminal plasma proteins.

Aspartate aminotransferase (AspAT) is present in seminal plasma of yellow perch, and its activity is about 0.25 % that seen in spermatozoa (Dabrowski at al. 1996). It can potentially be used as an indicator of sperm quality, since leakage of AspAT from spermatozoa into seminal plasma due to sperm damage can be measured.

Lactic dehydrogenase (LDH) has been identified in seminal plasma of Eurasian perch (Lahnsteiner et al. 1995). The activity of sperm LDH has been employed to demonstrate the cytotoxic effect of gossypol on yellow perch spermatozoa (Ciereszko and Dabrowski 2000).

Major oxidant defensive enzymes are also identified in Eurasian perch seminal plasma, including catalase, glutathione reductase, methionine reductase, peroxidase, and superoxide dismutase (Lahnsteiner and Mansour 2010). Treating semen with catalase results in substantial improvement of spermatozoa motility and membrane integrity.

Other enzymes described in seminal plasma include proteases, alkaline and acid phosphatases, ATPase, β-glucoronidase, butyryl cholinesterase, and glucose-6-phosphate dehydrogenase (Lahnsteiner et al. 1995; Kowalski et al. 2003, 2004).

5.2.3 Lysozyme Activity and Immunoglobulins

Lahnsteiner and Radner (2010) demonstrated the presence of the lysozyme N-acetylmuramide glycanohydrolase and immunoglobulins in seminal plasma of Eurasian perch (1–2 U L−1). Both proteins are important components of the defense system and are related to quality of fish sperm.

6 Spermatozoa Motility

Similar to most other freshwater and marine fish, spermatozoa in percids are immotile in the sperm duct due to osmolality of the seminal plasma (Lahnsteiner et al. 1995; Alavi et al. 2007). This contrasts with the mechanism of spermatozoa quiescence in Salmonidae and Acipenseridae, in which K+ is the main inhibitor of sperm activation in the seminal plasma (Cosson 2010; Alavi et al. 2012).

Initiation of spermatozoa motility in percids is triggered at discharge into hypo-osmotic freshwater (Lahnsteiner et al. 1995; Alavi et al. 2007; Boryshpolets et al. 2009). Studies report greater than 80 % spermatozoa motility in Eurasian perch (Lahnsteiner et al. 1995; Alavi et al. 2007, 2010; Henrotte et al. 2010; Lahnsteiner 2011), walleye (Satterfield and Flickinger 1995a; Bergeron et al. 2002; Casselman et al. 2006), and pikeperch (Křišťan et al. 2014). The initial spermatozoa motility rate does not differ among percids and cannot be used as a species indicator. Potential for motility in fish sperm is a hormone-dependent mechanism regulated by the hypothalamus-pituitary-gonad axis leading to increase of intracellular pH and cAMP in spermatozoa during maturation of spermatozoa in the sperm duct (Miura et al. 1992). In percids, as in other fish species (Alavi et al. 2009, 2012), waves propagate along the flagellum starting immediately upon initiation of sperm motility, while later in the motility phase waves are restricted to the region proximal to the head and becoming absent at the end of the motility period (Alavi et al. 2010) (Fig. 5.2). After initiation of motility, the percent of motile spermatozoa, velocity, and flagellar beat frequency decrease in Eurasian perch (Fig. 5.3) and pikeperch (Fig. 5.4). Decrease in these parameters is chiefly due to depletion of intracellular ATP required for axonemal beating (Fig. 5.3). Duration of spermatozoa motility in percids ranges from a few seconds to a few minutes and is reflected in inter-individual differences within a species and inter-specific differences among Percidae. In Eurasian perch, most studies report duration lasting less than 120 s after activation in freshwater, distilled water, or saline medium (Fig. 5.3) (Lahnsteiner et al. 1995; Alavi et al. 2007, 2010; Rodina et al. 2008). In walleye, the spermatozoa motility period is less than 120 s after activation in freshwater (Satterfield and Flickinger 1995a; Casselman et al. 2006; Green and Kelly 2008). In pikeperch, the duration of sperm motility is reported as 5–9 min (Korbuly et al. 2009) or 1–2 min (Křišťan et al. 2014) after activation in freshwater (Fig. 5.4). Recently, Lahnsteiner (2011) reported duration of sperm motility in Eurasian perch longer than 2 h following activation in a medium composed of 75 mM NaCl, 2 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 20 mM Tris, pH 8.0 (210 mOsmol kg−1). The observed differences in spermatozoa velocity and duration of motility among species may be related to initial ATP content. Duration of sperm motility shows a positive relationship to spermatozoa ATP content. Green and Kelly (2008) reported a longer period of motility in walleye spermatozoa containing a greater amount of ATP. Initial spermatozoa velocity also differs among percids. Values are reported as 122 μm s−1 in Eurasian perch (Lahnsteiner et al. 1995; Alavi et al. 2007, 2010), 94–120 μm s−1 in walleye (Casselman et al. 2006), and 158–165 μm s−1 (Křišťan et al. 2014). Spermatozoa velocity depends primarily on axonemal beating (flagellar beating frequency and wave parameters) and the morphology of the spermatozoa such as head size as well as the presence of a fin-like structure along the flagellum, which enhances flagellar beating (Alavi et al. 2009; Cosson 2010; Gillies et al. 2012).

Fig. 5.2
figure 2

Dark field stroboscope illuminated flagellum images of Eurasian perch (Perca fluviatilis) spermatozoa at 15, 30, 45 and 60 s post-activation in (a) buffered distilled water, 20 mM Tris, pH 8.0, (b) NaCl 50 mM, Tris 20 mM, pH 8.0 (c) sucrose 100 mM, Tris 20 mM, pH 8.0 and (d) NaCl 50 mM, CaCl2 1 mM, Tris 20 mM, pH 8.0. Semen was firstly diluted in NaCl 200 mM, NaHCO3 2.38 mM, pH 7.5 (osmolality 380 mOsmol kg−1) and motility was observed under (for details see Alavi et al. 2007). Beating of flagella were observed either in ionic or non-ionic activation media. (Arrows), Damage to flagella including blebs and loops. (In motile spermatozoa, flagellar wave are seen, while immotile spermatozoa can be recognized by straight non-moving flagellum.) (e) Eurasian perch spermatozoa beating 20 s post-activation in tap water (22 °C). Time interval between each image is 4 ms. The flagellum propagates 3 bends or 1.5 sine wave (High-speed video, Olympus i-speed TR 1000 frames/s)

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Fig. 5.3
figure 3

Changes in (a) spermatozoa motility, (b) velocity, (c) beating frequency of flagellum and (d) ATP contents in Eurasian perch (Perca fluviatilis) with time post-activation in buffered distilled water (DW), NaCl 50 mM, Tris 20 mM (NaCl), sucrose 100 mM, Tris 20 mM (sucrose) and NaCl 50 mM, CaCl2 1 mM, Tris 20 mM (CaCl2), pH 8.0–8.5 (Alavi et al. 2007, 2010; Hatef et al. 2010, 2011). Semen was diluted in NaCl 200 mM, NaHCO3 2.38 mM, pH 7.5 (osmolality 380 mOsmol kg−1). Spermatozoa motility and velocity are higher after activation in ionic or non-ionic activation media with 100–150 mOsmol kg−1 compared to those of buffered distilled water. Adding Ca2+ to the activation medium increase spermatozoa velocity

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Fig. 5.4
figure 4

Changes in (a, c) sperm motility (b, d) velocity in pikeperch (Sander lucioperca) post-activation in 45 mM NaCl, 5 mM KCl, 20 mM Tris, pH 8.5 (NaCl), 100 mM sucrose, 20 mM Tris, pH 8.5 (Suc) or 100 mM sucrose, 1 mM CaCl2, 20 mM Tris, pH 8.5 (Suc+Ca). (a, b) fresh spermatozoa; (c, d) spermatozoa incubated 24 h on ice. Addition of Ca2+ to the activation medium increase motility and velocity of incubated spermatozoa (c, d). No pre-dilution was performed in these observations

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7 Mechanism of Initiation of Spermatozoa Motility

Numerous factors contribute to initiation of spermatozoa motility in freshwater fish. The effects of pH, ions, and osmolality are critical to understanding the triggers of activation following sperm release from the genital pore into freshwater (Alavi and Cosson 2005, 2006; Morisawa 2008) and can provide valuable information for development of effective sperm immobilization and activation solutions that can be used for development of protocols for short-term storage, cryopreservation, and artificial insemination at fish farms (Ciereszko et al. 2000).

7.1 Effects of pH on Sperm Activation

Motility of Eurasian perch spermatozoa has been demonstrated to be induced upon dilution in a buffered NaCl solution (100 mOsmol kg−1) at pH 5.5–9.0 (Lahnsteiner et al. 1995; Hatef et al. 2011). The percent motile spermatozoa at pH 7.5 is not significantly different from that at pH 5.5, but decreases have been demonstrated at pH higher than 9.0. Lahnsteiner et al. (1995) reported lower spermatozoa velocity after activation in an acidic (121 μm s−1 at pH 6.5) or alkaline condition (119 μm s−1 at pH 9.0) compared to that of pH 7.5 (174 μm s−1). Therefore, pH is not the key factor regulating spermatozoa motility in seminal plasma or after release into the aquatic environment. Seminal plasma pH has been reported as 8.3 in Eurasian perch (Lahnsteiner et al. 1995).

7.2 Effects of Ions and Osmolality on Initiation of Spermatozoa Motility

The initiation of spermatozoa motility appears to be ion-independent, as motility can be activated in both ionic (NaCl or KCl) and non-ionic (sucrose or glucose) solution (Fig. 5.5) (Lahnsteiner et al. 1995; Alavi et al. 2007, 2008b). Spermatozoa motility is suppressed with dilution in 150–175 mM NaCl or KCl (300–350 mOsmol kg−1) (Fig. 5.5a), and sucrose or glucose at 300 mM prevents spermatozoa motility in Eurasian perch (Fig. 5.5b), demonstrating that osmolality is the principal inhibitor of spermatozoa motility in the seminal plasma of percids. Upon dilution of sperm in NaCl, KCl, or sucrose-based solution, the initial motility rate is usually not different from that in distilled water, but decreases at osmolalities above 200 mOsmol kg−1. The highest spermatozoa velocity is reported at 100 mOsmol kg−1 (Lahnsteiner et al. 1995; Alavi et al. 2007, 2008b). Therefore, media containing NaCl or KCl or sucrose at 100 mOsmol kg−1 and media of 300–350 mOsmol kg−1 are suggested as activation and immobilizing media for percid sperm, respectively (Fig. 5.6).

Fig. 5.5
figure 5

Effects of osmolality on spermatozoa motility and velocity in Eurasian perch (Perca fluviatilis) after activation in (a) NaCl or (b) sucrose based activation media. Osmolality higher than 300–350 suppresses initiation of spermatozoa motility

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Fig. 5.6
figure 6

Changes in (a) sperm motility, (b) velocity, (c) ATP contents and (d) ultrastructure in Eurasian perch (Perca fluviatilis) following 24 h incubation in NaCl 200 mM, NaHCO3 2.38 mM, pH 7.5 (osmolality 380 mOsmol kg−1) (Hatef et al. 2011). Spermatozoa motility and velocity activated in 50 mM NaCl, 20 mM Tris, pH 8.5 (110 mOsmol kg−1) decrease following incubation which are associated with decrease in ATP content and damage to flagella and mitochondria

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Spermatozoa velocity in Eurasian perch and pikeperch increases when calcium (Ca2+) is added to the activation medium, but the percent motile spermatozoa does not differ (Alavi et al. 2007; Křišťan et al. 2014) (Figs. 5.3 and 5.4). This is evidence of involvement of Ca2+ in axonemal beating, and further studies using specific Ca2+ channel blockers to investigate the role of Ca2+ in the initiation of spermatozoa motility in percids is essential.

In extremely low osmolality activation media, such as buffered distilled water, blebs appear along the flagellum that prevent correct and efficient wave propagation (Alavi et al. 2007) (Fig. 5.2). Later in the motile period, the tip of the flagellum becomes curled into a loop, shortening its length and affecting sperm velocity (Fig. 5.2). Similar damage has been observed in other freshwater fish and is probably the primary reason for the rapid decrease in sperm motility and velocity after sperm activation under very low osmolality conditions (Alavi et al. 2009).

8 Factors Affecting Semen Quality

As in other teleosts (Ciereszko 2008; Alavi et al. 2008a), numerous factors influence semen quality in percids (Ciereszko 2008). Season, photothermal regime, hormonal stimulation, nutrition, rearing conditions, status of males, age, and semen contamination have been studied in percids.

8.1 Season

Reproduction of temperate fish, including percids, is clearly affected by the season (Wang et al. 2010). In yellow perch, semen volume increases from January to April (Dabrowski et al. 1994). Before the spawning season (November-January), the semen of Eurasian perch is characterized by high spermatozoa concentration (59–66 × 109 cells mL−1) as compared to 36–45 × 109 cells mL−1 in February and April (Alavi et al. 2010). This is associated with decrease in seminal plasma osmolality from a range of 423–523 mOsmol kg−1 in November and January to 373–292 mOsmol kg−1 in February and April (Alavi et al. 2010). These changes are likely related to the semen hydration phenomenon, a physiological characteristic at the final stage of testicular development that leads to increase in semen volume (Ciereszko 2008). Along with physiological changes with spermatozoa maturation, spermatozoa motility increases from the beginning to the middle of the spawning season, and subsequently decreases at the end of the spawning season, likely due to aging of spermatozoa (Dabrowski et al. 1996; Król et al. 2006; Alavi et al. 2010).

8.2 Photothermal Regimes

Photoperiod and water temperature are basic environmental factors regulating reproduction of temperate fish. Phase-shifted photothermal cycles are frequently used to regulate time of sexual maturation and spawning in yellow perch (Dabrowski et al. 1996; Ciereszko et al. 1998) and Eurasian perch (Migaud et al. 2004, 2006). These studies showed that the shortened photothermal cycle accelerates testicular development, while continuous light inhibits it. Wang et al. (2008) determined that light intensity increases semen production.

8.3 Hormone Stimulation

As in most finfish with importance in aquaculture (Alavi et al. 2008a), hormone treatment has been applied to stimulate release of semen and/or synchronize spawning in percids. LHRHa, carp pituitary extract, or human chorionic gonadotropin (hCG), and a combination of follicle stimulating hormone (FSH), luteinizing hormone (LH), and metoclopramide have been evaluated for yellow perch, Eurasian perch, and pikeperch, respectively (Dabrowski et al. 1996; Kucharczyk et al. 1996, 1998; Zakęś 2007; Zakęś and Demska-Zakęś 2009). However, the success of spermiation induction may be associated with reproductive season. For example, more effective stimulation by LHRHa has been reported in yellow perch in February and March compared to April (Dabrowski et al. 1996).

8.4 Nutrition and Antinutritional Factors

Most information concerning effects of nutrition on reproductive performance of males involves essential fatty acids and antioxidants. Lee and Dabrowski (2004) studied effects of supplemental ascorbic acid and α-tocopherol and demonstrated potential transfer of both vitamins from to the testis of yellow perch, resulting in an increase in semen quality. Henrotte et al. (2010) showed that the composition of spermatozoa fatty acids is highly correlated with dietary fatty acid composition. However, no effects have been detected on semen quality. Considering the function of unsaturated fatty acids in metabolism and cryo-resistance of spermatozoa, further studies are required to investigate whether supplemented fatty acids can improve spermatozoa quality in percids. Wang et al. (2008) determined that the initial nutritional status of fish (together with light intensity) is important to increase semen quality in Eurasian perch.

Plant-derived ingredients of fish meal contain numerous anti-nutritional substances, such as gossypol, which is present in the cotton plant and has been shown to have male contraceptive activity. In vitro studies have demonstrated that gossypol inhibits spermatozoa motility and LDH activity in yellow perch, leading to decrease in fertilization ability (Ciereszko and Dabrowski 2000). These results suggest a potential for reproductive impairment in fish when cottonseed-containing diets or organic fertilizers are used in perch aquaculture.

8.5 Rearing Conditions

Cejko et al. (2008) reported higher spermatozoa motility in pikeperch maintained in tanks in photothermal controlled conditions compared to those in ponds or cages. The photothermal regime has been shown to highly influence sperm quality in percids (Król et al. 2006).

8.6 Status and Age of Broodfish

Three status-dependent mating tactics have been identified for Eurasian perch according to body size: sneaking (small males), group spawning (midsized males) and dominant spawning (large males) (Wirtz and Steinmann 2006). In this classification, larger males produce spermatozoa superior to that of smaller males. Korbuly et al. (2009) also observed that large healthy pikeperch males generally produce more semen than smaller specimens. Therefore, size of broodfish could be considered a biological indicator in percids, since the number of spermatozoa and their swimming velocity are critical indicators of fertilization success in walleye (Casselman et al. 2006). Ciereszko et al. (1998) found 3-year-old males to have higher spermatozoa concentration compared to 2-year-old males, and that these differences could be influenced by the photothermal regime.

8.7 Semen Contamination

Contamination of semen by urine is a serious threat to spermatozoa in percids. Studies show low initial spermatozoa motility in percids, particularly in pikeperch (Bokor et al. 2007; Cejko et al. 2008). This can result from spontaneous contamination of semen by urine during stripping, altering the osmolality of the seminal plasma. Semen contamination by urine not only influences spermatozoa motility but can result in decrease of spermatozoa fertilizing ability and affect success of cryopreservation (Linhart et al. 2003).

9 Short-Term Storage of Semen

Preserved semen can be useful in aquaculture to overcome problems such as shortage of males when females are ripe and can eliminate the need for transport of adult fish. For instance, walleye males are usually in short supply at the end of the spawning season, as their sexual maturation period is shifted 7–14 days in advance of females (Satterfield and Flickinger 1995b). Development of protocols for short-term storage is critical to ensuring a reliable source of semen and also provides a convenient means of increasing genetic diversity in hatcheries (Cloud et al. 1990). Essential steps in short-term storage of semen are to dilute semen in an extender as soon as possible after stripping, to store diluted semen in an oxygen-rich environment with the semen layer not exceeding 2–5 mm in thickness, and to maintain a temperature of 1–5 °C (Satterfield and Flickinger 1995a). Use of an extender provides ion concentrations and osmotic pressure at levels isosmotic to the seminal plasma, preventing initiation of spermatozoa motility (Rodina et al. 2004), protecting spermatozoa from osmotic damage and contaminants such as urine, and maintaining an ATP source required for flagellar beating as well as fertilizing ability (Linhart et al. 2003). At low temperatures, spermatozoa metabolism is reduced (Rurangwa et al. 2004). However, prolonged cool storage conditions can greatly affect the quality of semen, since anaerobic conditions and associated potential microbial contamination (possibly prevented by addition of antibiotics) may reduce spermatozoa motility and viability (Wayman 2003).

For walleye semen, three immobilizing media have been introduced as extenders (Moore 1987; Brown and Moore 1996; Malison and Held 1996; Satterfield and Flickinger 1995a): (a) CaCl2 0.117 g, MgCl2 0.134 g, Na2HPO4 0.236 g, KCl 1.872 g, NaCl 6.578 g, D-glucose 10.0 g, citric acid monohydrate 0.10 g, KOH (1.27 g/100 mL) 10 mL and bicine (5.3 g bicine/100 mL) 20 mL, pH 9.0; (b) NaCl 8.760 g, 5000 units of penicillin and 5 mg streptomycin per mL of 0.9 % NaCl 10 mL, pH 7.6; and (c) CaCl2 0.103 g, MgCl2 0.220 g, Na2HCO3 0.235 g, KCl 2.558 g, NaCl 5.780 g, pyruvate 6.0 g, citric acid monohydrate 0.1 g, KOH (1.27 g/100 mL) 10 mL, pen-strep 10 mL and HEPES 2.380 g, pH 9.0. Following 14 days incubation, more than 50 % spermatozoa were motile for 50–60 s in all three extenders. Different semen to extender ratios have been also evaluated, with no differences observed in fertilization rate of fresh semen and that preserved in each of the three extenders following 2–10 days storage with addition of antibiotics. Further studies have shown that antibiotics might not be necessary, since the semen contains natural antibiotics (Brown and Moore 1996).

Hatef et al. (2011) reported decrease in Eurasian perch spermatozoa motility from 99 % to 67 % and decrease in spermatozoa velocity from 228 to 175 μm s−1 following 24 h incubation of semen in an extender composed of 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, and 2.38 mM NaHCO3, adjusted to pH 8.5 (340 mOsmol. Kg−1) at ratio 1:50 (sperm:extender). Neither spermatozoa motility nor velocity decreased following 3 and 6 h incubation compared to fresh semen. Decrease in spermatozoa motility might be caused by damage to the plasma membrane, a key element for receiving hypo-osmotic signals required for activation of spermatozoa. Further analyses showed that the decrease in spermatozoa velocity might be related to depletion of ATP content as a source of energy for axonemal dynein ATPase activity, which has been also associated with damage to the fine structure of sperm, particularly mitochondria. Further studies are required to investigate fertilizing ability of spermatozoa in the above-mentioned formulated extender during short-term storage.

In pikeperch, Korbuly et al. (2009) studied incubation of semen using various immobilizing media and observed more than 50 % of spermatozoa were motile for over 120 s when the semen was diluted in Ringer’s solution (309 mOsmol. kg−1) or phosphate buffered saline (295 mOsmol kg−1) after 3 h incubation compared to fresh semen. Non-diluted semen, stripped manually from pikeperch, can be stored for up to 48 h at 4 °C (Křišťan et al. 2014). During period of storage, both spermatozoa motility and velocity decrease (Fig. 5.5), which is associated with the decrease in ATP content and damage to spermatozoa morphology and fine structure. In pikeperch, short-term semen storage has been investigated using two extenders: (a) 180 mM NaCl, 2.68 mM KCl, 1.36 mM CaCl2, 2.38 mM NaHCO3, pH 8.0 (343 mOsmol kg−1) and (b) 180 mM NaCl, 2.68 mM KCl, 2.38 mM NaHCO3, pH 8.0 (380 mOsmol kg−1). Results showed higher spermatozoa motility and velocity in diluted sperm compared to non-diluted sperm. Both non-diluted semen and semen diluted in an extender exhibited higher spermatozoa motility and velocity during the period of storage when 1 mM Ca2+ was added to the activation medium (Křišťan et al. 2014).

10 Sperm Cryopreservation

Cryopreservation is a method for long-term storage of viable spermatozoa in a frozen state in liquid nitrogen (−196 °C). Sperm cryopreservation provides biological advantages to conserve threatened fish, to reduce genetic diversity, and to increase effectiveness of artificial reproduction. Cryopreservation may provide valuable spermatozoa for late-ripening females, for crossbreeding fish from different populations, and for crossbreeding strains that mature at different times. Cryopreservation success is highly variable and depends on factors including biological characteristics of broodfish and physico-chemical conditions of cryopreservation: composition of cryoprotective extender, sperm to extender ratio, and freezing and thawing rates. Since sperm physiology, and particularly seminal plasma composition, varies widely among species, cryopreservation methods should be designed and/or evaluated for each species (Ciereszko et al. 2000; Kopeika and Kopeika 2008).

According to available data (Table 5.3), the first successful percid semen cryopreservation was achieved by Moore (1987) in walleye and by Ciereszko et al. (1993) in yellow perch. In both cases, multi-component cryoprotective extenders and a two-step freezing process in which sperm was initially cooled on the surface of dry ice were used (Table 5.3). Simplified extenders, i.e. ion-, sucrose-, or glucose-based solutions, have been used by Bergeron et al. (2002) and by Bokor et al. (2007, 2008) using pellet-freezing and floating-frame methods, respectively. Results showed higher numbers of frozen-thawed spermatozoa with a floating-frame method following dilution of semen in simplified extenders. Several studies of semen of various percids showed no significant difference between dimethyl sulfoxide (DMSO) and methanol (MeOH) as cryoprotectants (Table 5.3). These studies also indicated that cryoprotectants could be applied in the concentration range 6–10 % without significant effects on fertilizing ability of spermatozoa after thawing.

Table 5.3 Sperm cryopreservation in Percidae
Full size table

Procedures for cryopreservation of percid sperm can be summarized as follows: (1) sperm uncontaminated with urine is required, (2) sperm should be diluted 1:1–15 with a simplified ionic- or organic-based solution or extenders, (3) 6–10 % MeOH or 10 % DMSO can be used as cryoprotectant, (4) semen suspension in cryoprotective medium should be drawn into 0.25 or 0.5 mL straws prior to freezing, and (5) straws should be placed on a 3 cm thick floating styrofoam frame in liquid nitrogen for 3–10 min prior to deep freezing. Most frequently used thawing conditions are to place straws in a 40 °C water bath for 13 s; however, the thawing condition also depends on the volume of straws.

11 Conclusions

Although spermatozoa morphology seems to be similar within Percidae, studies of species belonging to this family will provide valuable information on inter-species differences in the ultrastructure, which is associated with sperm motility. In this context, the position of proximal and distal centrioles, number of mitochondria, flagellum length, and the presence of a fin-like structure are important.

The composition of seminal plasma in Percidae is well documented. Since seminal plasma is critical to spermatozoa viability and to protect against damage caused by microbes, xenobiotics, and oxidative stress, future investigations are required to characterize the function of seminal plasma compounds that contribute to protection against sperm aging in the reproductive system, adverse effects of endocrine disrupting chemicals, and effects of nutrients and anti-oxidants. It is also important to determine whether seminal plasma can be considered as an extender for cryopreservation and/or for in vitro maturation of spermatozoa.

Sperm motility in Percidae is induced by a hypo-osmotic signal that probably mediates Ca2+-dependent axonemal beating. In this context, it is important to understand the role of specific Ca2+ channels in the sperm membrane and Ca2+-dependent proteins in the molecular structure of the axoneme. Available literature shows that media with 100–200 and 300 mOsmol kg−1 can be considered potent activating and immobilizing solutions, respectively. Further studies on the effects of externally supplemented ATP, Ca2+, and anti-oxidants will provide valuable information for development of activation and immobilizing media for short-term storage and cryopreservation. ATP content has been investigated in the sperm of several percid species, but studies of other percids can identify inter-species differences in duration of sperm motility and velocity corresponding to ATP content and/or flagellum length.

This literature review shows that semen quality can vary in response to numerous factors and often reflects variability in environmental and handling conditions as well as individual and social conditions. Among environmental factors, season seems to be the most significant, and phase-shifted photothermal cycles can be developed to regulate sexual maturation and spawning season. In aquaculture, maintenance and nutrition can affect the quality of semen. Contamination is a serious threat to semen quality. Expanded knowledge of factors contributing to sperm quality is necessary to allow improvement of the control of reproduction.

Further studies are required to define the effects of parameters involved in sperm quality on success of cryopreservation. In this context, it is important to study how fatty acids, vitamins, and anti-oxidants in the diet may influence sperm cryo-resistance.