Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)
Introduction
Fish spermatozoon is usually differentiated into a head, a midpiece and a flagellum with the typical cylindrical arrangement of “9+2” microtubules. The head contains the nucleus and therefore the paternal DNA material. Energy required for sperm motility originates from mitochondria, located in the midpiece. Beating of the flagellum itself led to motility of the spermatozoon [1], [2], [3]. It has been already demonstrated that the family-, species- and subspecies-specific differences regarding the fine structure of spermatozoa can be related to functionality (motility and fertility) [1], [2], [3], [4], [5] and the physiological/biochemical characteristics of spermatozoa [6], [7], [8].
Fish spermatozoa are immotile in the testis [9], and acquire the potential for motility during transfer from the testis to the sperm duct [10], [11]. It is already well known that the two main factors of seminal plasma preventing the initiation of sperm motility are: its high potassium (K+) concentrations (in Salmonidae [12], [13], [14] and in Acipenseridae [15], [16], [17], [18]) and its osmolality, either low relative to external medium, which occurs in most marine fishes or high relative to freshwater in non-marine fishes [4], [19], [20], [21]. During natural reproduction, fish spermatozoa become motile after discharge into the aqueous environment due to external factors, such as low K+ concentrations in salmonids or acipenserids, and hypo- or hyper-osmotic shock in freshwater or marine fishes, respectively [22], [23], [24], [25]. Cellular studies on the mechanism of sperm motility in fish showed that spermatozoa use external factors as the triggering factor for initiation of the intracellular cascade of events that leads to the initiation of flagellar beating [22], [23], [26]. Sperm motility is generated by a highly organized microtubule-based scaffolded structured called the axoneme [27], [28].
Because motility of sperm in fish is influenced by several external factors such as pH, temperature, ions and osmolality [8], [22], [24], [25], [29], understanding effects of these factors are helpful in improving methods of artificial reproduction and provides information for developing better short- and long-term storage (cryopreservation) conditions for sperm [29], [30], [31]. In addition, it leads to comparative knowledge regarding species-specific differences in motility of sperm [4], [19], [25].
Specific studies on sperm motility in terms of flagellar beating and wave parameters have led to a better understanding of the internal mechanics explaining how the movement characteristics of sperm flagella are established [32]. For this purpose, fish spermatozoa are extremely suitable because they have remarkable variations in both structure and function [3], [4], [5], [32]. Most knowledge on sperm movement developed by simple flagella comes from studies on sea urchin (echinoids) sperm [33] which are long term swimmers (tens of hours). Nevertheless, fish spermatozoa show several original features: (1) their duration of motility is very short [34], (2) their ability to immediately initiate motility upon contact with the external medium, (3) the specific differences which are observed between freshwater and marine species [22], [23], [35] and (4) the variety in flagellar motility patterns exhibited among species and/or conditions [36], [37]. The flagellar movement of fish spermatozoa may be classified into two groups according to sperm structure [32]; (1) spermatozoa having elongated mitochondria and (2) spermatozoa having a simple structure with rudimentary mitochondria. The first and second type appears in fish using internal and external fertilization strategies, respectively.
The northern pike (Esox lucius L.) is a freshwater species inhabiting the northern hemisphere which has been cultivated extensively in Europe and Asia since the middle ages [38]. Males and females become sexually mature at age 2–3 and 3–4 years, respectively, and spring spawning occurs in the shallow waters when the temperature of this water reaches 4–7 °C [39], [40]. A few studies have been published on sperm biology in pike. These studies have shown that the stripped sperm density ranges 7–22 × 109 spermatozoa ml−1 and the total number of spermatozoa is 10–15 × 109 [41], [42], [43]. Moreover, the ionic composition and osmolality of seminal fluid are Na+ (116 mM), Cl− (116 mM) and K+ (25 mM), Ca2+ (1 mM) and 273 mOsmol kg−1 (44.45). Pike spermatozoa have a very short duration of motility, up to a maximum of 90 s in freshwater at 4 °C (temperature of activation medium) [42], [43], [44], [45], [46].
In the present study, the main objective was to investigate the effects of osmolality on: (a) sperm morphology during activation, (b) percentage of motile sperm and (c) sperm velocity. Various flagellar wave parameters (wave length, wave amplitude, number of waves and tracks curvatures) were also determined as a function of the osmolality of the activation media.
Section snippets
Sample collection
During the spawning season in March in Vodnany, Czech Republic, 16 mature male pike (total length 50–67 cm; body weight 912–1400 g) were captured from large natural ponds. After transportation to the hatchery, sperm samples were collected by abdominal massage. Hormonal induction was not used for sperm maturation. All attempts were made to avoid contamination of sperm by urine, mucus or water during stripping. Sperm samples were collected in syringes and kept in an ice box (0–2 °C) during
Sperm morphology
The northern pike spermatozoa are uniflagellated and are differentiated into a head without acrosome, a midpiece with a cylindrical shape and a tail region called a flagellum presenting one lateral fin (Fig. 1). Table 1 shows inter-specimen differences of the morphological parameters measured by SEM.
Osmolality effects on sperm morphology
Observations of spermatozoa after activation in distilled water using dark-field (Fig. 2a), phase contrast microscopy (Fig. 2b) and SEM (Fig. 2c) showed that, almost 30 s after triggering of sperm
Sperm morphology
In Esociformes, only the spermatozoa of E. lucius [47], chain pickerel (Esox niger) [48] and muskellunge (Esox masquinongy) [49] have been briefly described, but morphological parameters have not been quantitatively determined. In all three species, spermatozoa are uniflagellated, acrosomeless and have clearly differentiated head, midpiece and flagellum. Similar to E. masquinongy, the E. lucius spermatozoon have a spherical head, 1.40 μm in diameter. The midpiece is elongated in both pike and
Acknowledgements
This study was supported by USB RIFCH No: MSM 6007665809, GACR No. 524/06/0817 and IAA608030801. The authors appreciate funding support received from FAPEMIG (CVZ APQ 2578-5-04/07), Brazil for Ana Viveiros. The authors warmly thank Martin Psenicka and Martina Tesarova for their helps during preparation of samples for SEM and Ivana Samkova and Marie Pcena for their technical assistants.
References (65)
Fish gamete preservation and spermatozoan physiology
- et al.
Objective analysis of sperm motility in the Lake sturgeon (Acipenser fulvescens): activation and inhibition conditions
Aquaculture
(1997) - et al.
Biology of sperm and artificial reproduction in carp
Aquaculture
(1995) - et al.
Sperm motility in fishes. I. Effects of pH and temperature
Cell Biol Int
(2005) - et al.
Sperm motility in fishes. II. Effects of ions and osmolality
Cell Biol Int
(2006) - et al.
Semen of Perca fluviatilis L.: sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility
Theriogenology
(2007) - et al.
Optimization of artificial propagation in European catfish Silurus glanis L.
Aquaculture
(2004) - et al.
Cryopreservation of tench, Tinca tinca, sperm: sperm motility and hatching success of embryos
Theriogenology
(2007) - et al.
Stimulation of spermiation and induction of ovulation in pike (Esox lucius)
Aquaculture
(1980) - et al.
Ultrastructure of spermatozoa of tench Tinca tinca observed by means of scanning and transmission electron microscopy
Theriogenology
(2006)