WWW.WAS.ORG • WORLD AQUACULTURE • SEPTEMBER 2013 47 Brief Description of the Fish Sperm Cell Spermatozoa of teleost fishes showing internal fertilization have a simple structure (Fig. 1). Spermatozoa of carp Cyprinus carpio, as described by Billard (1970), represents an example of the primitive type. The head (2.5 µm diameter) is almost spherical. The midpiece has centrioles (base of flagellum) plus a few mitochondria (energy providers). The acrosome, a structure located at the front of the sperm cell and involved in the sperm/ egg binding process, is usually absent in teleost fish spermatozoa. In the case of sturgeons and paddlefish (chondrosteans), the head is elongate and possesses an acrosome, which is an exception. The axoneme has a typical arrangement of nine pairs of peripheral microtubules and one pair of central tubules. The system of microtubules in the flagellum represents the backbone of the motile apparatus of a sperm cell (Fig. 2). Each double microtubule carries two arms, which consist of ATPase micromotors called dynein. Through hydrolysis of ATP, dynein arms interact with its adjacent double tubule, causing sliding (Gibbons 1981). Because of the presence of interconnecting elements between peripheral microtubules, continuous sliding between them creates a tension and results in oscillations of the flexible flagellum (Satir 1984). The flagellum compartment is considered quite independent of the head. Activation of Sperm Motility For successful fertilization, sperm must reach, bind and penetrate an egg. Activation of sperm motility is the prerequisite for implementation of these processes. Fish spermatozoa stored in seminal plasma are immotile during transit of the genital tract of most externally fertilizing teleosts and chondrosteans. Motility is induced immediately after release of spermatozoa from the male genital tract into the aqueous environment. The nature of the external signal that triggers motility initiation is highly dependent on the fish reproduction environment (fresh- or seawater) and peculiarities of reproductive behavior. Seawater contains a high concentration of solute or ionic salts whereas freshwater has a very low solute concentration. The term osmolarity refers to the value of the total concentration of solutes irrespective of the chemical nature of each solute. Osmolarity is a main way by which spermatozoa motility is controlled. Measuring Motility Characteristics of Fish Spermatozoa Just after activation by the surrounding medium, sperm flagella of many fish species beat at a high frequency (up to 100 Hz) but for a short period (30 sec to several minutes), depending on species (Cosson et al. 2008 a,b, Cosson 2010). The initial velocity of sperm cells is high (up to 300 µm/sec), which makes observation and recording especially difficult (Cosson et al. 2008a). Several methods have been developed to study motility characteristics — including velocity of spermatozoa, percentage of motile cells and flagellar wave shape and its propagation — of these fast-moving sperm cells and to visualize flagella. These methods use acquisition of images of tracks followed by sperm heads or, alternatively, high-speed images of moving flagella in vivo. Today these methods use video techniques and, in some cases, allow the recording of frames using stroboscopic illumination at frequencies up to 800 Hz (Fig. 3). Computer-assisted processing of the frames or, more simply, of head tracks (CASA) allows comparison of parameters of the movements of fish spermatozoa exposed to (CONTINUED ON PAGE 46) FIGURE 3A. Normal propagation of waves in a sturgeon spermatozoon (from left to right). Sperm flagella show first and second waves of similar amplitude. Note elongated head with changes in orientation. Flagellum length = 55 µm, Flash frequency = 150 Hz. Images are every 0.05 sec. Initial video was at 750 frames/sec. FIGURE 3B. Wave patterns observed by high-speed video microscopy (phase contrast optics, 100 x lens). (a) Initiation of waves and normal wave propagation with damping in sturgeon sperm flagella. Sperm motility was triggered by transfer of sperm into a swimming solution containing 10 percent DMSO. (b) Fully activated sperm (21 s after mixing). Wave amplitude is increasing slightly from head to tip. Images are every 0.0107 msec. Initial video at 750 frames/sec. (c) Later stages of the motility period (36 s after activation). Sperm flagella still show progressive motility but are slower due to damping of waves from base to tip. Such damping is also observed in absence of DMSO. Images are every 0.0027s. Initial video at 1000 frames/sec. Bar = 10 μm. Photo from video by Galina Prokopchuk.
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