Spermatology in Aquatic Species

**11**

**Chapter 2**

*Jacky Cosson*

**1. Introduction**

**Abstract**

Fish Sperm Physiology: Structure,

For reproduction, most fish species adopt external fertilization: their spermatozoa are delivered in the external milieu (marine- or freshwater) that represents both a drastic environment and a source of signals that control the motility function. This chapter is an updated overview of the signaling pathways going from external signals such as osmolarity and ionic concentration and their membrane reception to their transduction through the membrane and their final reception at the flagellar axoneme level. Additional factors such as energy management will be addressed as they constitute a limiting factor of the motility period of fish spermatozoa. Modern technologies used nowadays for quantitative description of fish sperm flagella in movement will be briefly described as they are more and more needed for prediction of the quality of sperm used for artificial propagation of many fish species used in aquaculture. The chapter will present some applications of these technologies and the information to which they allow access in some aquaculture species.

**Keywords:** flagellum, sperm energetics, sperm signaling, sperm motility, osmolarity

The main function of a spermatozoon is to convey the male genome remotely to the female one, which occurs in case of fish by swimming in the external milieu, marine or freshwater. Spermatozoa must access, bind, and penetrate an egg, for successful fertilization. Therefore, most of the physiological activity of fish spermatozoa is motility oriented. These processes include as a prerequisite the activation of spermatozoon motility. In case of fish species, spermatozoa stored in the seminal plasma are immotile during transit through the genital tract of most externally fertilizing teleost and chondrostean. Motility is induced immediately following the release of spermatozoa from the male genital tract into the aqueous environment. External trigger agents for the initiation of motility depend on the species' reproductive behavior that is mostly controlled by the aquatic environment (fresh or salt water). Triggering signals include osmotic pressure, ionic and gaseous components of the external media, and, in some cases, egg-derived substances used for sperm guidance. Environmental factors influencing fish spermatozoon motility have received a large attention: these extensive studies led to several mechanisms of activation for freshwater and marine fish spermatozoa. However, after reception of the signal, a transduction pathway initiated by these mechanisms must lead the information to the flagellar motility apparatus (axoneme). This review presents

Factors Regulating Motility, and

Motility Evaluation

#### **Chapter 2**

## Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation

*Jacky Cosson*

#### **Abstract**

For reproduction, most fish species adopt external fertilization: their spermatozoa are delivered in the external milieu (marine- or freshwater) that represents both a drastic environment and a source of signals that control the motility function. This chapter is an updated overview of the signaling pathways going from external signals such as osmolarity and ionic concentration and their membrane reception to their transduction through the membrane and their final reception at the flagellar axoneme level. Additional factors such as energy management will be addressed as they constitute a limiting factor of the motility period of fish spermatozoa. Modern technologies used nowadays for quantitative description of fish sperm flagella in movement will be briefly described as they are more and more needed for prediction of the quality of sperm used for artificial propagation of many fish species used in aquaculture. The chapter will present some applications of these technologies and the information to which they allow access in some aquaculture species.

**Keywords:** flagellum, sperm energetics, sperm signaling, sperm motility, osmolarity

#### **1. Introduction**

The main function of a spermatozoon is to convey the male genome remotely to the female one, which occurs in case of fish by swimming in the external milieu, marine or freshwater. Spermatozoa must access, bind, and penetrate an egg, for successful fertilization. Therefore, most of the physiological activity of fish spermatozoa is motility oriented. These processes include as a prerequisite the activation of spermatozoon motility. In case of fish species, spermatozoa stored in the seminal plasma are immotile during transit through the genital tract of most externally fertilizing teleost and chondrostean. Motility is induced immediately following the release of spermatozoa from the male genital tract into the aqueous environment. External trigger agents for the initiation of motility depend on the species' reproductive behavior that is mostly controlled by the aquatic environment (fresh or salt water). Triggering signals include osmotic pressure, ionic and gaseous components of the external media, and, in some cases, egg-derived substances used for sperm guidance. Environmental factors influencing fish spermatozoon motility have received a large attention: these extensive studies led to several mechanisms of activation for freshwater and marine fish spermatozoa. However, after reception of the signal, a transduction pathway initiated by these mechanisms must lead the information to the flagellar motility apparatus (axoneme). This review presents

 the current knowledge with respect to (1) membrane reception of the activation signal and its transduction through the spermatozoon plasma membrane via the external membrane components such as ion channels or aquaporins; (2) cytoplasmic trafficking of the activation signal; (3) final steps of the signaling, including signal transduction to the axonemal machinery, and activation of axonemal dynein motors and regulation of their activity; (4) signaling involved in guidance processes that control the sperm/egg approach and meeting; and (5) pathways supplying energy for the short flagellar motility period of fish spermatozoa. For each step in this signaling process, quantitative methods were developed to evaluate the quality of the sperm samples that are used for aquaculture propagation of many fish species. These methods as well as examples of their usefulness for application to fish artificial reproduction are presented in the last part of this chapter.

#### **2. Fish sperm structure and spermatogenesis**

The main traits of the flagellar mechanics of spermatozoa and the main factors that regulate their motility have been described in details in a recent review chapter by Cosson et al. [1].

#### **2.1 Structure of fish spermatozoa: a brief presentation**

 Compared to mammalian spermatozoa, the structure of a fish spermatozoon is qualified as "simple sperm", mostly because the flagellum structure does not include additional columns flanking the motor part (axoneme) that are present in mammal sperm. In teleost fish, spermatozoa generally have no acrosome (in contrast to chondrostean such as sturgeon), and the impenetrable chorion presents a micropyle that gives access to the membrane of the oocyte. Spermatozoa often show a spherical nucleus with homogenous, highly condensed chromatin, a nuclear fossa, a midpiece of variable size with or without a cytoplasmic channel, and one or two long flagella [2]. Moreover, fish spermatozoa can be classified into two forms, aqua sperm and intro-sperm, according the external or internal mode of fertilization, respectively [2].

The main components present in a fish spermatozoon [3, 4] are: the head is occupied mostly by the nucleus with paternal DNA material. In most fish species, the head has an almost spherical shape (diameter of 2–4 μm). In some case such as sturgeon, paddlefish, and eel spermatozoa, the shape of head is elongated (up to 9 μm long and 2 μm wide) [5–7], the midpiece is mostly composed of the centrioles, and the mitochondria (usually from 2 to 9 per each spermatozoon) is generating energy (ATP) for motility [8]. In a mature spermatozoon, because the protein synthesis machinery is absent, no gene expression occurs. The centriolar complex of midpiece consists of the proximal and the distal centrioles, which forms the basal body of the flagellum, used for anchoring the flagellum to the head of the sperm cell.

The flagellum is a highly conserved organelle during evolution, and there are very few differences between molecular composition of sperm flagella relatively to that in protists [1, 10]. Fish sperm flagellar length varies from 20 to 100 μm, depending on species. Flagellar bending is generated by a highly organized cylindrical system of microtubules, called the axoneme, emanating from the basal body [11]. In turn, the canonical "9 + 2" axoneme consists of nine pairs of peripheral microtubular doublets and one central pair of singlet microtubules. This structural arrangement [12] is illustrated in **Figure 1**. Such axonemal pattern is highly conserved and almost identical among eukaryotic cilia and flagella from protozoans to human. Nevertheless, in Anguilliformes and Elopiformes sperm flagella present a "9 + 0" pattern lacking central microtubules [6, 7, 13].

*Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

#### **Figure 1.**

*Schematic structure of the head-tail junction of a model spermatozoon: the artistic view applies to a teleost such as trout [9] as an example of sperm cell. m, membrane surrounding both the head (top) and the tail; a, axoneme, the mechanical part actuating the flagellum (bottom); t, microtubule doublets, the major scaffold of the 9 + 2 axoneme; c, centriolar complex, at the basal part of the axoneme, made of microtubule triplets. In many fish species, the mitochondria (not shown for simplification) are localized in this head-tail junction zone, and two centrioles are orthogonally assembled to form the centriolar complex.* 

 The structural connections between the nine peripheral outer doublets and the sheath surrounding the central pair occur through the radial spokes. The central pair of singlets is enclosed in this sheath of proteins forming a series of projections that are well positioned to interact with each of the spoke heads and regulate the wave propagation [14]. Each of the outer doublets is connected to adjacent pairs of doublets by nexin links, presenting elastic properties allowing to resist the free sliding of the microtubules; nexin is a dynein regulatory protein [15]. The peripheral doublets are strung with two rows of dynein arms along the entire length of microtubules. These dynein arms consist of macromolecular ATPase complex [16, 17] and represent the basic motor actuating the whole axoneme; they extend from an outer doublet toward an adjacent doublet [18]. Both the spokes and the dynein complex contain different calcium-binding proteins so as for flagella to be able to respond to regulation by free calcium concentration through altering their beating pattern [19, 20]. As briefly described above, axonemes are complex structures composed of at least 500 different protein components [21].

The bending process in an axoneme is caused by sliding between two adjacent doublets of outer microtubules that slide relatively to each other due to the motive force, generated by molecular dynein motor activity [16]. Due to enzymatic hydrolysis of ATP by the latter, which induces force generation of the power stroke of individual dynein molecules, the dynein arms interact with tubulin of the B tubule

 from the adjacent doublet, causing a process of active sliding in a cooperative way [22]. Several local bending processes occur because this sliding activity is present in only some segments of the axoneme at a given time, while other segments remain inactive [4]. Wave propagation from head to tip provokes the translation of the whole spermatozoon in the opposite (forward) direction.

#### **2.2 Spermatogenesis in fish**

For obtainment of full efficiency of motility, all the elements of the flagellum must have been, during spermatogenesis, correctly assembled mostly as an elongated structure called axoneme playing the role of propelling engine, surrounded by the plasma membrane, and this device must be provided in energy in terms of ATP [8], the fuel common to many cell types that is mostly generated by mitochondrial respiration in case of fish sperm as detailed in paragraph 4.

Spermatogenesis is an important phase in case of fish spermatozoa because it is the ATP store that constitutes the main source of energy that will sustain the short but highly energy-demanding motility period [8].

A detailed description of fish spermatogenesis is well documented in many species. Briefly, spermatogenesis is a developmental process during which a small number of diploid stem cells (spermatogonia) produce a large number of highly differentiated spermatozoa carrying a haploid, recombined genome, and a structurally complete flagellum. Survival and development of those germ cells depend on their close contact with specific cells called Sertoli cells. Apart from their phagocytic role, the Sertoli cells change the growth factor expression, and subsequently, modulate germ cell proliferation/differentiation via complex mechanisms involving, in fish, both pituitary gonadotropins LH and FSH that stimulate gonadal sex steroid hormone production directly by the activation of another cell type called Leydig cells.

Fishes represent the largest and most diverse group of vertebrates. However, our knowledge on spermatogenesis in this group is limited to a few species used in basic research and/or in aquaculture biotechnologies such as guppy, catfish, cod, eel, medaka, salmon, tilapia, trout, and zebrafish. In an amniote vertebrates (fishes and amphibians), one observes a cystic type of spermatogenesis, which presents two main differences compared to higher vertebrates [23]. First, within the spermatogenic tubules, cytoplasmic extensions of Sertoli cells form cysts that envelope a single, clonally and hence synchronously developing group of germ cells deriving from a single spermatogonium. Second, the cyst-forming Sertoli cells retain their capacity to proliferate also in the adult fish. Sertoli cells are surrounding and nursing one synchronously developing germ cell clone. Different clones being in different stages of development generate a tubular compartment containing differently sized groups of germ cells in different stages of spermatogenesis.

 So as for the distribution of spermatogonia in the germinal compartment, one can observe either a first type of restricted spermatogonial distribution, which is found in the higher teleost groups, such as in the order Atheriniformes, Cyprinodontiformes, and Beloniformes, where the distal regions of the germinal compartment are occupied by Sertoli cells surrounding early, undifferentiated spermatogonia. While the cells divide and enter in meiosis and the cysts migrate toward the region of the spermatic ducts located centrally in the testis, this is where spermiation occurs, i.e. the cysts open to release spermatozoa.

In a second type, where an unrestricted spermatogonial distribution that is considered a more primitive pattern found in less evolved taxonomic groups, such as in the order Cypriniformes, Characiformes, and Salmoniformes, occurs, spermatogonia are spread along the germinal compartment throughout the testis. The cysts do not migrate during their development. In addition, intermediate forms also

#### *Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139*

exist between restricted and unrestricted spermatogonial distribution, such as in Perciformes, tilapia, Pleuronectiformes, or Gadiformes.

 Therefore, the development of spermatogenic cells strictly depends on their interaction with the somatic elements of the testis, among which Sertoli cells play a crucial role. During fish spermatogenesis, Sertoli cells are formed by mitosis just in time and in exactly the number required. This tailored Sertoli cell proliferation was first described in the guppy [24]. So far, we observe that spermatogenesis is a highly organized and coordinated process, in which diploid spermatogonia proliferate and differentiate to form spermatozoa in their final morphology. The duration of this process is usually shorter in fish than in mammals. In principle, this process can be divided, from a morpho-functional point of view, in three different phases: (i) the mitotic or spermatogonial phase with the different generations of spermatogonia, (ii) the meiotic phase with the primary and secondary spermatocytes, and (iii) the spermiogenic phase with the haploid spermatids emerging from meiosis and differentiating, without further proliferation, into flagellated spermatozoa.

 Analysis of the role of hormones reveals a complex process. Three steps at which reproductive hormones play a critical regulatory role are (i) the balance between selfrenewal and differentiation of spermatogonial stem cells, (ii) the transition from type A spermatogonia to rapidly proliferating type B spermatogonia, and (iii) the entry into meiosis. During later developmental stages, on the other hand, the endocrine system seems to ensure a permissive rather than stimulatory role, enabling Sertoli cells and possibly other somatic cells to generate a microenvironment that germ cells require to proceed through meiosis and spermiogenesis [25]. In case of fish, three types of spermiogenesis have been described, based on the orientation of the flagellum relatively to the nucleus and on whether or not a nuclear rotation occurs.

#### **2.3 Flagellum genesis**

The main organelle in the flagellum, the axoneme, is resulting from the progressive assembly of groups of elements synthesized in the cell body and then transported to the flagellar compartment and delivered to the flagellar tip to elongate it thanks to the motility of specific transporters called intra-flagellar transport (IFT) along the internal side of the flagellar membrane [21]. The trafficking is ensured by two important molecular motors, belonging to the dynein group (retrograde, meaning from tip to base of the flagellum) and to the kinesin group (anterograde, meaning from the cell body to the flagellum tip) [26].

#### **3. Successive steps leading to fish sperm motility**

#### **3.1 The maturation step**

Maturation is the step following the end of spermatogenesis and that provides to the spermatozoon its ability to respond to motility-activating factors [27]. Signals for maturation are quite various among fish species. Sperm maturation is also regulated by the endocrine system.

 Examples of sperm maturation have been studied in details in different fish species: salmonids, cyprinids, and sturgeons. In salmonids, the group of Morisawa, in Japan, demonstrated, in particular in case of salmon, that maturation is mainly under control of cAMP and pH of the water where fish are transiting during migration [28]. In carp, results from Redondo et al. [29] indicate that an ionic equilibration across the sperm membrane is the main factor responsible for maturation. In case of sturgeon species, it was shown that spermatozoa are not able to become

activated at simple contact with external water [30]; sperm cells need a transient contact with urine, the latter getting mixed with milt prior to ejaculation [31] which is enough to render spermatozoa fully motile.

#### **3.2 The activation step per se**

 This is a very brief event lasting a fraction of a second [32, 33]. Among several kinds of activating signals, one can mention (1) osmolarity, the most common, (2) specific ions such as K<sup>+</sup> in a few species (salmonids and sturgeons as examples), and (3) other signaling molecules such as CO2 in few cases [34]. The osmolarity signal depends on the external medium where fish delivers its sperm: marine fish spermatozoa activate when coming in contact with sea water, a salty solution of high osmolarity, while freshwater fish species shed their sperm in a very low-osmolarity medium. In both cases, the spermatozoa sustain a large stress that consists in a jump from seminal fluid ranging an osmolarity 300 mOsmol/kg to either sea water (around 1100 mOsmol/kg) or freshwater (maximum 50 mOsmol/kg).The environmental osmotic pressure appears consistently to be the main factor involved in fish motility activation among species [35, 36]. In a few fish species, osmolarity is acting in synergy with another factor such as specific ions [35]. In some marine species such as herring, activation of spermatozoa requires egg-derived substances. Two types of sperm-activating factors have been identified in Pacific herring, *Clupea pallasii*, eggs: a water-soluble protein released into the surrounding water [37] and a water-insoluble sperm motility-initiating factor localized in the vicinity of the micropylar opening of eggs [38].

#### **3.3 The perception of the signal by the sperm membrane**

 Freshwater fish sperm cells when released into the surrounding water can increase their cytoplasmic volume in response to osmotic stress. In case of carp spermatozoa, the cell volume increases several times as a result of the influx of water [39]. Results of Cabrita et al. [40] show that small change of cell volume occurs in response to hypo-osmotic shock. The comparative study of Bondarenko et al. [41] puts forward a large species specificity regarding the osmotic reaction (swelling) among freshwater species and demonstrates that there is no change of trout sperm volume measured during the motility period. Altogether, the volume change, if any, represents a long-term osmotic reaction rather than the immediate signal for motility activation given its time delay (several tens of seconds) relative to the briefness of motility appearance (less than a second according to Prokopchuk et al. [33].

 A series of experiments by Takei et al. [42] aims to better explore the respective roles of K+ ions and osmolarity; this paper proposes a mechanism involving aquaporins and volume changes as a response to osmolarity stress. However, the volume changes measured by the authors are of low amplitude: the engendered volume difference is so low that it cannot be responsible of a physiological role in motility control. Furthermore, the time scale and the volume change measurements obtained at 5 min after motility activation correspond to time scales that are not fit with the previously published results of Prokopchuk et al. [33] showing that the motility response occurs about 100 ms after reception of the activation signal of fish spermatozoa. In addition, previous results by Bondarenko et al. [41] demonstrate that no significant volume change follows the motility activation of trout spermatozoa, a situation that contrasts with that of carp.

In many marine teleost species, hypertonicity induces the motility of spermatozoa. Nevertheless, an increase in external osmolality is sometimes not the only condition for motility activation of marine fish spermatozoa: in case of herring, this *Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

activation also needs the contact of sperm with egg-derived substances, facilitating fertilization [37, 38, 43, 44]. Sperm-activating factors are two types of in the Pacific herring, *Clupea pallasii*: a water-soluble herring egg protein [37, 43, 44] and waterinsoluble initiating factor, from the vicinity of the micropylar opening of the egg [38]. In another group of marine fish species collectively named flatfishes, the main signal perceived by the sperm membrane is the CO2 concentration [34] that is high in the genital tract but very low in sea water. The intracellular equilibrium between CO2 and bicarbonate constitutes the second step in the control of intracellular ionic concentration that leads to regulation of flagellar motility [33].

#### **3.4 The transduction of the signal across the membrane and the cytoplasm**

 In fish spermatozoa, external signals triggering sperm motility activation are acting at the level of spermatozoon plasma membrane, hyperpolarization/depolarization of membrane, and ion channels or aquaporins activity, but this topic is still challenging because of the scarcity of experimental results in this area. As mentioned above, it is clear that water transport itself is not a main process involved in fish sperm motility activation. Nevertheless, in the presence of aquaporins in the head and flagella plasma membrane of the seawater fish, gilthead sea bream (*Sparus aurata*) spermatozoa and their involvement in cAMP-mediated phosphorylation of axonemal proteins were established [45, 46]. In this species, the water efflux via aquaporins would determine a reduction in the cell volume, which would raise the intracellular concentration of ions. This would lead to the activation of adenylyl cyclase and motility initiation by cyclic AMP-dependent protein phosphorylation and dephosphorylation [46]. Such cascade of events remains hypothetical because of the timing of such process compared to the extreme briefness (less than 0.1 s) of the reaction of the axoneme activation [33].

The presence of different ion channels was described in sperm plasma membrane [34]. Cytosolic pH could be considered as another participant of signaling pathways, as it is known to be one of the parameters influencing sperm motility [47, 48]. Environmental conditions that inhibit spermatozoa motility can decrease the intracellular pH, resulting in a more acidic cytoplasm in nonmotile spermatozoa than in motile spermatozoa [49]. The decrease of internal pH in sperm would directly affect flagellar movement through inhibition of dynein activity. The involvement of the Na+/H+ exchangers in sperm motility activation process was reported for *Cyprinus carpio* [50] and was proposed for *Brycon henni* [51]. The former authors suggest that the regulation of the exchangers depends on osmolality conditions [52]. According to Krasznai et al. [53], an opening and closing of K+ channels in the plasma membrane of the spermatozoon under hypo-osmosisinduced initiation of sperm motility is resulting in a remarkable local hyperpolarization or depolarization of the spermatozoon plasma membrane. Such transient depolarization may open Ca2+ channels, resulting in an influx of Ca2+ and activation of the flagellar motility of carp sperm.

The involvement of K<sup>+</sup> and Ca2+ transport through ion channels at the plasma membrane of spermatozoa in the triggering of the motility initiation has also been shown for rainbow trout (*Salmo gairdneri*) spermatozoa [54]. As for Na+ channels, Tanimoto and Morisawa [54] supposed that Na<sup>+</sup> channels do not play an important role in sperm motility in rainbow trout, although they did not exclude its possible involvement in sperm motility control, for example, through Na+ -H+ exchange.

Altogether, the precise mechanisms of regulation of ion channel activity and their participation in the hyperpolarization of the spermatozoon membrane, which is associated with the activation of sperm motility [35, 53], remain poorly understood. What are the next-step processes occurring at the level of membrane leading the subsequent activation of the axoneme?

#### **3.5 Transduction and reception of the signal at the axoneme level**

 Among other processes, an increase of intracellular concentration of ions could lead to the activation of adenylyl cyclase, which in turn would determine the motility initiation by a cAMP-dependent protein phosphorylation and dephosphorylation mechanism [46]. It is known that, in mammals, protein tyrosine phosphorylation of several proteins is upregulated by reactive oxygen species (ROS). ROS (especially H2O2) may enhance tyrosine phosphorylation through the selective suppression of tyrosine phosphatase activity [55] or activation of adenylyl cyclase, thus producing a higher cAMP level and leading to the subsequent activation of the serine/threonine kinase A [56].

Cyclic AMP is an important factor in the activation process of fish spermatozoa. The link between cAMP concentration increase and motility initiation at the axoneme level was mainly investigated in Salmonidae. It involves a complex series of phosphorylation and dephosphorylation events. This includes the cAMP-dependent phosphorylation of the 15 kDa movement-initiating phosphoprotein [57, 58] of a PKA [59] and of the 22 kDa dynein light chain [60]. Protein phosphorylation is also regulated by proteasomes [60, 61]. The precise dependence between protein phosphorylation and microtubule sliding and movement initiation is still under investigation. A Ca2+-mediated and/or cAMP-dependent phosphorylation signaling mechanism through the radial spoke/central pair system of the axoneme has been proposed [62, 63].

The potential role of reactive oxygen species (ROS) generated at the contact of sperm with aerobic condition such as the external medium at ejaculation and the molecular mechanisms by which these reactive metabolites exert their biological activity has been put forward by Baker and Aitken [64]. A gas, NO, was also observed to enhance motility of fathead minnow spermatozoa [65]. Nevertheless, the mechanism by which NO affects sperm motility is probably unrelated to osmolality, because of its very low active concentration range.

#### **3.6 The motility period**

During the period after spermatozoa comes in contact with an activating medium, the ion concentrations inside the sperm cell are rebalanced, and osmotic pressure affecting the sperm membrane becomes harmful for sperm integrity, limiting the period of motility to a short interval [32]. These phenomena are much faster and more obvious in freshwater species, in which sperm motility usually does not last for more than 0.5–2 min [32].

**Video 1** can be viewed at https://vimeo.com/310085381.

Brook trout (*Salvelinus fontinalis*) spermatozoa recorded while swimming in a swimming medium composed of low osmolarity (10 mM Tris at pH 8.0 + 10 mM CaCl2). Remark the briefness of motility (around 30 s duration at 10°C) (courtesy of Dr. Galina Prokopchuk).

In case of sperm collection for artificial reproduction or in vitro studies, particular care should be taken to avoid precocious activation of motility: one should avoid any contact of sperm cells with external water [66] or urine during stripping [67, 68]. Any assessment of motility parameters should be started as soon as possible after sperm activation, bearing in mind that the earliest period of motility (the most efficient one) should be characterized. Fish spermatozoa are usually characterized by a very high initial velocity (up to 200 μm s<sup>−</sup><sup>1</sup> ) due to the high flagellar beat frequency (up to 100 Hz; [32]); this "most active" period lasts only a few seconds immediately after contact with the activating medium.

Values of all motility parameters decrease rapidly immediately after initiation of fish sperm flagella movement [36, 69], which is why any motility parameter must

*Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

 refer to a precise time point after activation for intra- or interspecies comparisons [36, 69, 70]. Generally, the duration of motility is a trade-off between the level of energy stocks possessed by a cell and the process of osmotic damage experienced by this cell. The latter is more critical in freshwater fish species, and the former is important for marine fish [36]. At a precise time point, most spermatozoa have very similar characteristics [69, 70].

**Video 2** can be viewed at: https://vimeo.com/310086859.

Carp (*Cyprinus carpio*) spermatozoa recorded while swimming in two different media successively; the first part of the record shows the carp sperm population activated in a swimming solution composed of 45 mM NaCl +5 mM KCl + Tris at pH 8.0, while the second part shows spermatozoon activation in a low-osmolarity medium (distilled water + Tris at pH 8.0). Remark the curling of the flagellar tip which limits the duration of motility at low osmolarity (courtesy of Dr. Volodymyr Bondarenko).

#### **4. Energetic of sperm motility**

Storage of energy mostly results from mitochondrial respiration that generates ATP. Energy metabolism also involves other compounds such as creatine phosphate that contributes to the maintenance of the intracellular energy level in connection with ATP.

#### **4.1 Mitochondrial respiration**

In many fish species, measurement of respiratory activity presents difficulties because of the low oxygen consumption of spermatozoa, in contrast to model species such as sea urchin [71]; in addition, the low respiratory activity remains almost unchanged when fish spermatozoa are transferred into motility-activating solutions [72], while it is about 50-fold increased when sea urchin sperm is transferred into sea water [71]. Efficient respiration needs to be coupled in mitochondria to ATP production via the ATP synthase [73]. For estimation of the full respiratory capacity of mitochondria, it is useful to apply diffusible "uncouplers" such as CCCP or FCCP (carbonylcyanide-4-trifluromethoxy-phenylhydrazone): these compounds are diffusible through the membranes and allow full rate of electron transfer in the electron chain of mitochondria without restriction due to its control by ATP synthase. The effects of respiratory inhibitors such as oligomycin [73] or KCN and their relationship with ATP stores of fish sperm were studied in details by Dreanno et al. [74]. Mitochondrial inhibitors have little effect in case of trout [75–78] or turbot [79] spermatozoa. Respiration rate in quiescent fish spermatozoa (before motility activation) needs to be only minimal but enough to maintain this ATP level prior to ejaculation. Such low but substantial respiration is enough for basal metabolism to maintain ionic exchanges and balances across the plasma membrane [8, 76, 79].

#### **4.2 Generation and storage of ATP**

 Generation of ATP by mitochondria occurs by electron transfer along the mitochondrial respiratory chain that generates a proton gradient across the inner mitochondrial membrane. Dissipation of the proton gradient occurs by passage of H+ through a specific ATP synthase localized at the inner part of the mitochondrial membrane [8, 73]. ATP thus accumulated is transported out of the mitochondrion by a translocase (ATP-ADP exchanger) toward the flagellar compartment; then, ATP molecules diffuse along the axoneme possibly assisted by a carrier device called energy shuttle as explained in Section 4.4.

 The energy stored in fish sperm prior to and used during the motility period was evaluated in several fish species [80–82]. This was described in turbot, for example [79, 83, 84], sea bass [85] perch [86], bluegill [87, 88], trout [71, 89, 90], carp [39, 91], sturgeon [92, 93], and catfish [94]. Values for spermatozoa of other fish species can also be found in Cosson [8, 32, 95], Dzyuba et al. [30], and Ingermann [96].

#### **4.3 Consumption of ATP during the motility period**

ATP is the only high-energy compound that is hydrolyzed by the motor protein of the axoneme called dynein ATPase [16, 17, 97]. Dyneins are macromolecular assemblies of more than 1 MDa molecular weight linearly bound to each microtubule doublet of the axoneme and which function is mechanochemical. Its role it to collect the chemical energy generated by hydrolysis of an ATP molecule so as to induce a trans-conformation that constitutes the elementary step of movement generation. Rows of dyneins positioned along each microtubule amplify in a cooperative way this elementary movement so as to provoke the sliding between adjacent microtubules of the axoneme. Differential sliding generates bending, and the bending waves that propagate along the flagellum (usually from the head to the tip of the axoneme) result in a translational movement of the whole sperm cell [21].

In case of fish spermatozoa, dynein molecules have an intense activity: this fast activity results in a beat frequency (up to 80–100 Hz) much higher than in species other than fish. As a result, ATP is hydrolyzed at high speed, and the ATP store is rapidly decreasing, becoming partly exhausted at the end of the motility period [32].

#### **4.4 Other energetic molecules assisting ATP maintenance**

Following its synthesis by mitochondria, ATP should be transported and distributed all along the flagellum so as to supply chemical energy to sustain the mechanical energy generated by the dynein motors that are distributed all around the flagellar axoneme. Theoretical considerations lead to postulate the presence of a distribution system that ensures a constant ATP concentration at any point along the axoneme [98, 99]. Such shuttle system involves an additional high-energy compound and assistance of enzymatic system that was shown to be present in fish sperm cells [90] and is detailed below.

Even though the ATP molecule is the most common high-energy compound used as a fuel for many cell functions, including motility [8], several other highenergy molecules are present in living cells such as creatine phosphate or arginine phosphate. In fish, creatine phosphate (CrP) is the main high-energy compound that was characterized in spermatozoa of several fish species as a complement of ATP [74, 79, 85]. In the last decades, many studies have demonstrated the decrease of the ATP concentration inside the fish sperm cells during the motility period [39, 67, 74, 76, 79, 85, 90, 91, 93, 100]. A more restricted number of studies have investigated the concentration of ATP related compound such as ADP, AMP, CrP, and others [74, 79, 85, 100]. All these compounds are part of an intracellular network under control of different enzymes that are able to transfer high-energy phosphate bonds from one to another (see Figure XX in Chapter 1 of Cosson [21]), for example, the equilibrium ATP< = >ADP + Pi is catalyzed by enzymes called ATPases. In another example, ADP + CrP < = >ATP + Cr is controlled by enzymes called creatine kinases. One creatine kinase is mitochondrial, while a second one is in the flagellum and distributed all along the axoneme (**Figure 1**). The mitochondrial creatine kinase delivers CrP that diffuses along the flagellum, both creatine kinases being present in trout sperm cells. The rate of diffusion of CrP molecules is higher than that of ATP [99]. Such an arrangement of catalytic activities and substrates

*Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

**Figure 2.** 

*Evolution of the flagellar shape of fish spermatozoa during the motility period. From left to right, successive positions of a turbot (Scophthalmus maximus) spermatozoon video recorded at different time points postactivation: (a) right after transfer in sea water, (b) 2 min later, (c) more than 3 min later, (d) after full stop. Dark field microscopy with stroboscopic illumination (150 Hz); 100× objective lens [32].* 

constitutes an intracellular network ensuring the correct production and distribution of energy in fish sperm cells (**Figure 1**) and is called the "ATP shuttle" [99].

 A local axonemal paralysis appears in sperm flagella of many species during the motility period: this is an indication that the renewal of ATP is not fully efficient, and the production of ATP from CrP in this distal portion of the flagellum is too slow, while the proximal portion, that is close to the mitochondrion, can use directly the ATP still produced by the latter. Stiffening of the distal flagellar tip was observed in vivo (but not in reactivated sperm) in two sea urchin species [101] but also in trout sperm (Cosson, unpublished) after application of thiourea, an inhibitor of respiratory phosphorylation (**Figure 2**).

#### **5. Control of fish sperm physiology by other factors**

Changes in the environment of fish spermatozoa impose other chemicals as well as physical constrains: temperature is controlling sperm physiology at many levels such as membrane permeation, enzymatic activities, or energetic metabolism [102]. Fish species are exposed to a large variety of temperature conditions, especially during their reproduction period; thus, an optimal choice of temperature is a key parameter for controlling the best conditions for best adapted sperm physiology.

 Viscosity of the swimming medium is also an important factor influencing the physiology of fish spermatozoa. According to physical laws, especially microfluidics, progression of the sperm cell occurs because of the friction of the flagellum against the external milieu, water being a quite viscous fluid. Higher viscosity is encountered by fish spermatozoa when getting close to the fluids surrounding the egg, which modifies and thus controls the swimming physiology of the sperm cells [21, 103] including female cryptic choice of sperm cells by the egg [104].

Among other factors that play a signaling role for fish physiology are some molecules that control the sperm/egg interplay at fertilization; these molecules are commonly called chemoattractants and are able to finely tune the motility function of fish spermatozoa so as to optimally guide sperm cells to egg which ultimately results in an increase of fertility success [21]. In case of fish, the spermatozoon must localize the entrance point at the surface of the egg so as to penetrate and deliver the male genome [105]. Ultimately, the male genome will combine with that of the female so as to constitute the zygote.

#### **6. An update of modern technologies allowing fish sperm quality assessment**

#### **6.1 CASA systems**

 Evaluation of fish sperm swimming performances needs at first good quality video records so as to measure the distance covered by each sperm head for a time period corresponding, for example, to the time separating two successive video frames, such as 20 ms for the European video standard (**Figure 3**).

Several works in the last decade have reported the use of CASA systems to assess sperm motility in fish [106]. Based on the integration by the computer of successive positions of the moving head of spermatozoa in consecutive frames of video records to calculate the trajectories and their characteristics, CASA describes different parameters of sperm swimming linked to velocity, for example, VCL or instant speed (frame to frame displacement) along the real track, VAP or velocity along a smoothed track, VSL or progressive velocity following the straight line from the origin to the end of the track during the corresponding period of time and other parameters linked to the wobble of sperm head such as Mean angular displacement (MAD), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) and linearity. Lastly, the ratio between average path and straight-line path is used to describe the straightness of the trajectories (reviewed in Rurangwa et al. [107]).

The CASA system recently developed by Wilson-Leedy and Ingermann [108] as a plug-in to image J software freely available from NIH site (http://rsb.info.nih.gov/ ij/plugins/casa.html) has been tested in different species including zebrafish [109]. More recently, it was improved and adapted to trout sperm motility by Purchase and Earle [110].

One important aspect for motility estimation of sperm quality by CASA is that the practical conditions employed to perform such tests are in several respects not reflecting the natural situation. Many broadcast spawners like salmon or trout

#### **Figure 3.**

*Video image of swimming sturgeon spermatozoa. Dark-field video microscopy with stroboscopic illumination. Flashes are every 10 ms. At bottom right is the indication from the stop-watch giving the time (in milliseconds) spent since motility activation.* 

#### *Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139*

 reproduce in highly turbulent water, which certainly influences the sperm/egg meeting chances and thus the fertilization success. Effect of such turbulent water shear at some optimal values was studied using biophysical methods by Crimaldi and Browning [111], and it was shown experimentally to increase the proportion of fertilized eggs in sea urchin [112].

 Also, for practical reasons, CASA records are obtained in conditions where spermatozoa swim in the vicinity of glass surfaces: such situation was shown in literature to affect motility parameters [113, 114]. An important consequence of this is that the motility parameters mostly refer to a situation where sperm cells swim in a planar manner; it is well known since early studies [115] that when spermatozoa swim freely far from any surface, they adopt a helical trajectory. The latter was shown to decrease up to 10-fold the efficient gross velocity but surprisingly to increase around 6-fold the fertilization kinetics [116]. All these findings emphasize several limits of the application of these CASA systems when used to predict quality of sperm regarding the fertilization rate.

The comparative values of sperm velocity among fish species, including salmonid, can be found in Cosson [32] with respect to motility duration and ATP stores prior to activation.

#### **6.2 Evaluation of flagella performances**

 Behavior of the flagellum determines the motility guideline of the spermatozoon, so the description of intrinsic flagellar wave properties is considered as one of the most informative methods for assessing and controlling sperm motility. In order to observe the detailed pattern of live flagella or of their major components, it was proposed to use phase contrast or dark field optical microscopy with high magnification (40×−100×) objective lenses, which, if applied with oil immersion, result in a bright image of the very small diameter object that constitutes a flagellum. To achieve complementary assessment, additional methods, such as stroboscopic illumination or high-speed video techniques, allow to record sperm during its motion and specially to obtain flagellar images of high quality and resolution. Multiflash stroboscopic illumination thus allows visualization on each frame of well-defined successive positions of a same moving spermatozoon at time intervals ranging in milliseconds. Alternatively, high-speed video recording provides higher spatial and temporal resolutions (up to several 1000 images/s). Serial frames individually selected from such video records allow to follow successive positions (every millisecond or less) of flagellum waves covering one or several full beat cycles [32].

**Video 3** can be viewed at https://vimeo.com/309942086.

 Legend of the video: a sturgeon spermatozoon was recorded by phase-contrast video microscopy according to conditions similar to those described in **Figure 4** (courtesy of Dr. Bondarenko Volodymyr). Spermatozoa of Siberian sturgeon (*Acipenser baerii*) were activated by dilution in pond water and recorded 10 s after activation. The length of flagella is about 50 μm long. Image rate is 100-fold slower than real. Remark that some spermatozoa show abnormal shape.

Evaluation of sperm flagellum performances on a large variety of fish species leads to a series of predictions briefly summarized below (see **Figure 5**):


 While the whole cell is moving from left to right, the flagellum wave progresses from right to left. The wavelength is defined as the distance "L" between two inflection points. The half wave amplitude is represented as "A" and the bend angle of each wave as "a". The number of waves generated every second is called the beat frequency. Such parameters are quantified and used to characterize fish sperm cells exposed to various swimming situations affecting their physiology.

Two other examples of high-speed video records of fish spermatozoa are presented below.

#### **Figure 4.**

*Successive positions of a sturgeon sperm flagellum recorded by high-speed video microscopy. Initial record was at 5000 images/s with a 100× phase-contrast lens and an Olympus high-speed video camera. In this panel from left to right, successive images collected every millisecond are presented so as to show the wave propagation of three successive waves and the minor progression of the head tip (white straight lines) during this short time period.* 

#### **Figure 5.**

*Flagellar parameters of a swimming fish spermatozoon. The sperm cell, such as that recorded according to Figure 3, is represented in two successive positions (1–2) separated by a short time period (0.5 ms, for example).* 

*Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

**Video 4** can be viewed at https://vimeo.com/310100596.

Pangasius (*Pangasianodon hypophthalmus*) spermatozoa recorded by high-speed video combined with dark-field microscopy and was visualized 10-fold slower than normal. Sperm movement was recorded at 13 s post-activation in a 10% sea water solution. Length of flagellum is about 50 μm (courtesy of Dr. Galina Prokopchuk).

**Video 5** can be viewed at https://vimeo.com/310102833.

 Tilapia (*Sarotherodon melanotheron heudelotii*) spermatozoon recorded by high-speed video microscopy and visualized 20-fold slower. Sperm movement was recorded at 18 s post-activation in 50% sea water containing bovine serum albumin (BSA at 0.5%) to prevent sticking to the glass slide. Notice the short sperm flagellum in this species and the swollen midpiece, mostly composed of mitochondria (courtesy of Dr. Galina Prokopchuk).

#### **6.3 Biochemical methods**

#### *6.3.1 Respiration, oxidative stress, free radicals, and DNA damage*

Fish spermatozoa present a low respiratory rate that makes this evaluation quite delicate because of the sensibility of detection methods. Respiratory activity commonly uses oxygen electrodes that measure the oxygen concentration of a small volume of sperm suspension. These media are either preventing or activating motility and can be complemented by different effectors (substrates, uncouplers, or inhibitors) of respiration. In general, mitochondrial inhibitors have little effect on the motility of fish spermatozoa. Sperm oxygen consumption rate published in literature was presented in a comparative way for about 10 different fish species by Ingermann [96] and shows that values present a large variability from 1.4 to 70 nmoles O2/min/109 spermatozoa depending on species.

The contact of fish sperm with the external milieu occurring at ejaculation leads to exposure of sperm cells to high concentration of oxygen, provoking different kinds of stress [117]. Among other chemicals responsible of stress, reactive oxygen species are highly aggressive [118, 119]. The oxidative stress can be evaluated by several methods. Results of these studies show that several protections against the oxidative stress are present in fish sperm cells and in the seminal fluid [31, 120].

Also, the presence of free radicals leads to DNA damage during stressing situations such as those due to application of cryo-techniques that influence the quality of the progeny [121]. Recent studies on genes and protein expression of fish sperm cells show that both are controlled by various factors of the external milieu such as salinity or timing during the reproductive season [122, 123]. Among other factors, the level of phosphorylation of specific proteins constitutes important signaling factors controlling function efficiency of fish spermatozoa [124, 125]. Proteomics represent a promising approach to study specific physiological situations encountered by fish spermatozoa [126].

#### *6.3.2 Evaluation of energetic compounds concentration*

ATP content of sperm cells can be evaluated by several methods, including measurement in a single spermatozoon cell as recently shown by Chen et al. [127]. The most popular method for evaluation of the ATP content classically uses a coupling with the light-emitting system composed of luciferin and luciferase. A full evaluation of the storage of energy in fish sperm cells needs the determination not only of the internal content of ATP but also that of other energetic compounds that are

 able to exchange high-energy phosphate bonds able to be transferred to ADP and allow to reconstitute the intracellular ATP store. Such evaluation was established in case of sturgeon sperm [100, 128] by the use of liquid chromatography combined to HPRS or in case of turbot or sea bass sperm where the adenine nucleotides' energetic balance was determined by H<sup>+</sup> -NMR and 31P-NMR analysis, [74, 79] as well as in trout by 31P-NMR [89, 90]. All these results clearly point out to the fact that ATP level can be rescued by the CrP generated by the mitochondrial metabolism. This means that other phosphagen compounds are as important as ATP in the energy balance of fish sperm cells [129].

#### **7. Conclusion**

Fish sperm physiology is under control of various parameters of the external milieu: the latter is subjected to changes due to the different environmental conditions that sperm cells have to deal with such as (1) the ionic concentration of internal as well as external fluids, (2) the pH, (3) the osmolarity, (4) the temperature, and (5) specific molecules acting as signals such as chemoattractants that control the sperm-egg interaction at fertilization [21]. In fish spermatozoa, the interplay between the different actors results in a complex signaling network that exquisitely optimizes the various functions of fish spermatozoa, especially that of motility, in a large variety of situations. A better understanding of this complex network is important so as to decrease the effects of possible damage (osmotic, oxidative, etc.) when fish sperm cells are exposed to drastic conditions such as those imposed during application of cryopreservation methods.

#### **Acknowledgements**

The author is thankful to the Faculty of Fisheries and Protection of Waters, Univ. of South Bohemia in Ceske Budejovice, Czech Republic, especially to Dr. Galina Prokopchuck and Dr. Volodymyr Bondarenko for their high-speed video records of fish spermatozoa. The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic projects CENAKVA (LM2018099), by project Biodiversity (CZ.02.1.01./0.0/0.0/16\_025/0007370 Reproductive and genetic procedures for preserving fish biodiversity and aquaculture), and by the Grant Agency of the University of South Bohemia in Ceske Budejovice (125/2016/Z).

*Fish Sperm Physiology: Structure, Factors Regulating Motility, and Motility Evaluation DOI: http://dx.doi.org/10.5772/intechopen.85139* 

### **Author details**

Jacky Cosson University of South Bohemia, Ceske Budejovice, Czech Republic

\*Address all correspondence to: jacosson@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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### Section 3

## Climate Change and Aquatic Environment

**39**

**Chapter 3**

**Abstract**

Temperate Lakes

*Arne N. Linløkken*

Climate Change and Monitoring of

Provided the predicted 2°C temperature increase during this century, lake ecology will go through dramatic changes, and this must be addressed in fish management in purpose of exploitation as well as in species preservation. In temperate lakes with fish communities dominated by cold-water and cool water fish, temperature increase will affect the species dominance. Extended growth season will benefit recruitment of less cool adapted species, total fish density may increase and growth will decrease of some species. Lakes dominated by salmonid fish may become dominated by cyprinids and percids. Primary production will increase due to extended growth season and increased precipitation. This can reduce the oxygen level in the deep layer of lakes when the organic matter decomposes, whereas the upper layer is too warm for cold-water species. In addition, increased density of small plankton feeding fish will reduce the algae feeding zooplankton. Lakes should be monitored by means of modern and sophisticated methods, monitoring lakes from satellites and in situ loggers, and pelagic fish may be counted by echosounding. To counteract increasing density of plankton feeding fish, fish biomass removal

**Keywords:** pelagic zone, plankton, plankton feeders, predation, echo sounding,

Research on biological resources of any kind may be divided in two categories: research on economical important species to attain optimal exploitation strategy and research to reveal ecological roles of species, commonly in a conservation perspective. The first point is obviously linked to the second, whereas the second point's connection to economy may be unclear and absent, at least apparently. Aquatic organisms of economic importance are mostly found in marine environments [1], while the economical value of freshwater fisheries in most cases is small [2], due to small water bodies and consequently small amounts of potential yield, though there are exceptions, like in the big lakes in North America [3]. It is also still important for hunting and gathering people in some parts of the world [4]. Freshwater bodies are nevertheless important to human society for many purposes, for drinking water, irrigation, and bathing and as landscape elements [5]. The water quality is therefore important in several perspectives, and guidelines for monitoring are recommended by the European

is a possible measure, though the effect is limited in time.

electronic loggers, remote sensing

Commission [6], among others.

**1. Introduction**

#### **Chapter 3**

### Climate Change and Monitoring of Temperate Lakes

*Arne N. Linløkken* 

#### **Abstract**

Provided the predicted 2°C temperature increase during this century, lake ecology will go through dramatic changes, and this must be addressed in fish management in purpose of exploitation as well as in species preservation. In temperate lakes with fish communities dominated by cold-water and cool water fish, temperature increase will affect the species dominance. Extended growth season will benefit recruitment of less cool adapted species, total fish density may increase and growth will decrease of some species. Lakes dominated by salmonid fish may become dominated by cyprinids and percids. Primary production will increase due to extended growth season and increased precipitation. This can reduce the oxygen level in the deep layer of lakes when the organic matter decomposes, whereas the upper layer is too warm for cold-water species. In addition, increased density of small plankton feeding fish will reduce the algae feeding zooplankton. Lakes should be monitored by means of modern and sophisticated methods, monitoring lakes from satellites and in situ loggers, and pelagic fish may be counted by echosounding. To counteract increasing density of plankton feeding fish, fish biomass removal is a possible measure, though the effect is limited in time.

**Keywords:** pelagic zone, plankton, plankton feeders, predation, echo sounding, electronic loggers, remote sensing

#### **1. Introduction**

 Research on biological resources of any kind may be divided in two categories: research on economical important species to attain optimal exploitation strategy and research to reveal ecological roles of species, commonly in a conservation perspective. The first point is obviously linked to the second, whereas the second point's connection to economy may be unclear and absent, at least apparently. Aquatic organisms of economic importance are mostly found in marine environments [1], while the economical value of freshwater fisheries in most cases is small [2], due to small water bodies and consequently small amounts of potential yield, though there are exceptions, like in the big lakes in North America [3]. It is also still important for hunting and gathering people in some parts of the world [4]. Freshwater bodies are nevertheless important to human society for many purposes, for drinking water, irrigation, and bathing and as landscape elements [5]. The water quality is therefore important in several perspectives, and guidelines for monitoring are recommended by the European Commission [6], among others.

Water quality monitoring is concerned about water chemistry, physics, and biology. Certain elements are more important and are easy to monitor regularly, like phosphorus, nitrogen, acidity, clarity (Secchi depth, color, and turbidity), algae, and chlorophyll [6]. Optical instruments to analyze algae and chlorophyll concentrations are available, and a great benefit with the electronic measure devices is the possibility to install loggers for continuing monitoring [7]. Some biological elements are more laborious and expensive, like algae taxonomy, macro vegetation, zooplankton taxonomy, and fish abundance and ecology.

Lake ecology is affected by natural elements like local geology, climate, lake size, and bathygraphy [8], and a predicted climate change with a temperature increase of 2°C [9] during this century will provide serious consequences. In addition, human activity by the lake and in its catchment, even on remote places, affects the water quality. The organisms comprising a community may be more or less exclusive or rare, and some may be vulnerable or even threatened [10]. Ideally, there should be conducted genetic surveys on every species, but it would probably be a vast of resources. Nevertheless, species or populations that are considered as somehow vulnerable or unique should be analyzed by means of microsatellites or single-nucleotide polymorphism to describe the at-present state as a reference for future surveys [11–15]. Tissue samples should be preserved to be available for future analysis methods. The references are also useful for monitoring effective population size [13–15] and potential changes of allele frequencies, by random or as effects of natural selection.

#### **2. Lakes as ecological indicators**

Freshwater of lakes, rivers, and groundwater is important for all terrestrial life. There is scarcity of water in parts of the world [16], whereas in the temperate zone, it is usually available, though scarcity may occur in periods of the year. Water bodies are important sources of drinking water, and the quality is monitored for chemical and biological elements. Especially toxicants and pathogens are important, as they are mostly invisible by the eye. Other changes may be visual, like increased abundance of algae, planktonic, or on the bottom substrate or in fish nets. This may indicate eutrophication and could be serious. It indicates high levels of phosphorus, commonly the minimum factor in freshwater [8], though nitrogen also influences the primary production in lakes [17]. Increased phosphorus concentration may be added along lake shores or in the tributaries. Sources can be traced, and this could be a broken sewage pipe and runoff from a droppings pit or from fertilized fields resulting in an acutely polluted tributary. Algae bloom is a problem in hypertrophic lakes, and one characteristic trait is the cyanobacteria, among which, some may produce toxicants when occurring in high density [18, 19]. This has led to death of pasture cattle after drinking the water [20]. Even if such extremes are avoided, high algae production gain more biomass than the grazing chain from zooplankton to fish which may be consumed and decompose, and algae biomass is handled by the detritus chain in deepwater layer where oxygen deficit may occur [8]. Oxygen concentration at different depths is an important factor and is easy to measure with modern equipment. Oxygenated water precipitates iron (III) phosphorus and enriches the sediments, whereas oxygen deficit reduces the iron, and iron (II) phosphorus is soluble and is brought back into the water column during the spring mixing [8]. Phosphorus and chlorophyll a are therefore normally included in lake monitoring programs, and the chlorophyll a concentration is a good indicator of a lake's "health" condition [21]. Oxygen is very serious and may in worst case become

#### *Climate Change and Monitoring of Temperate Lakes DOI: http://dx.doi.org/10.5772/intechopen.84393*

chronic, and the lake sediments must be covered or replaced with other kinds of substrate, gravels and sand with low phosphorus content [22].

 The lake characteristics and the fish community are mutually dependent of each other, but the fish community also depends on accessibility for freshwater species. Freshwater organisms immigrated after the glaciations, and the migrations were hampered or stopped by the topography. Mountains and waterfalls make up effective obstacles to fish distribution, and the distribution of species was determined by the time of immigration and the time of land uplift that created the obstacles. The distance from the glacial refuges and the topography decided the possibility for the entrance. Some fish communities therefore have few species, unaffected of the lake environment. This is clearly demonstrated in Norway, where the river systems in the western part of the country, that is, west of the mountain range, almost exclusively harbor fish of anadromous origin, which means no cyprinids, percids, or pike (*Esox lucius*) [23]. Cyprinid species normally dominate in eutrophic lakes, when present [24–26], and if not, a lake can be eutrophic but with a simpler community. It makes a difference whether the dominating pelagic fish species are cyprinids, coregonids, or Arctic charr (*Salvelinus alpinus*) [27]. They can all feed on zoo-plankton, but with different efficiency, due to different body size, population density and not least, different density of the gillrakers that filter food items from the water. The dense gill rakers of most of the cyprinids filter small zooplankton species that slip through the gill rakers of coregonids, not to mention those of Arctic charr and brown trout (*Salmo trutta*) [28]. This again affects the zooplankton grazing capacity, as the most important herbivorous species are large and more catchable than the smaller species, and algae blooms become more probable [29, 30].

 Lakes may serve perfectly as indicators of environmental health condition of its surroundings and its catchment, and lakes are easily observable. Watercolor, clarity, and vegetation development can alarm the public in a lake's vicinity if dramatic changes occur. Bathing, fishing, or just observations from the shore may give a clue.

#### **3. Forming of lakes and their ecosystems**

 The occurrence of a lake demands a substrate tight enough to hold water, some kind of damming, natural or manmade (eventually built by beavers), and a water supply that exceeds evaporation. Tectonic processes, land uplift, landslides, volcanoes, and quaternary processes can create holes capable of holding water when filled [8]. On the Northern Hemisphere, the glaciers, until 10,000–12,000 years ago, caused land excavations that became lakes and moraines that could dam the water.

In glaciated areas, when the ice thawed, and a completely barren land appeared, the primary succession started, affected by the local geology, and minerals bound in rocks and stones were released by physical and chemical weathering. Algae, lichens, mosses, and plants started assimilating CO2 and, according to their needs, phosphorus, nitrogen, and other minerals [8]. Primary production was not hampered by organic bound nutrition and could flourish from the beginning. Grazers appeared and nutrition became in part bound in biota. Lakes got their sediments, slowly but steadily increasing, littoral macro vegetation developed, and animals, crustaceans, insects, mollusks, and others, appeared. Fish were more limited by waterways than most other aquatic organisms, like insects and small invertebrates that can be carried by other organisms or even by wind. In elevated areas, fish were not necessarily

 a result of the natural process but in many cases brought there by humans [31–33]. This was probably the first significant impact man had on lakes. Stools from sparse populations of Stone Age humans had probably a nonsignificant effect on lake productivity. Introduction of fish, which are mostly second (or higher)-order consumers, adds a predation pressure on organisms of several phyla, classes, and orders, among which, arthropods (especially crustacean and insects), annelids, mollusks, vertebrates, and, among those, other fishes, are important. If one fish species or five or more fish species are present, they structure the community through predation, depending on the species assemblage and the physical and chemical prerequisites of the lake. When the EU Water Directive demands lakes to be restored/maintained in "good status" [34], this is not unambiguous as it depends on the original state, that is, what species, nutrition load, acidity, and content of dissolved particles are and were present.

 Monitoring lakes should always be done by comparing the present state with an assumed original state, although a new state is not necessary disastrous. There are many examples of successful introductions of fish species, from a human point of view. What the original state was can to some extent be illuminated by means of sediment analysis, as organisms with scales, like planktonic crustaceans, are preserved [35, 36]. The state should be stable, if not in a mal state, and spring and autumn circulation turning the water column to supply the deep water with oxygen should take place regularly. That will keep the lake sound, so the detritus is treated effectively with oxygen present, and fish dying off under the ice is avoided, as well as bad smell from methane and hydrogen sulfide. Fishing and bathing are of interest for public, and this public use of environmental resources makes people conscious about their state.

#### **4. Monitoring of lake ecology**

 When monitoring fish communities, observations and measurements are conducted with a certain precision, and most importantly, the methods are described thoroughly enough to be repeated by others. Monitoring the lake ecology, one way or the other, may be regarded as testing a very wide/imprecise hypothesis: Is something changing? A more precise hypothesis can be formulated later, if the monitoring reveals that something in fact is changing. One species may become more abundant, whereas others become sparse, and an explanation can hopefully be found among the factors or variables that were monitored. At present, the temperature is a hot candidate. Others are nutrition load, acidity, new (alien) species, and eventually new parasites [37, 38]. The latter may become very harmful to indigenous species and populations [39].

 Important variables may be logged electronically and even be transferred wireless to data archives, and for the large-scale overview, remote sensing by means of satellite images is probably the optimal method [42]. The horizontal overview given by satellite images is superior to the traditional spot sampling. Images taken from planes may surpass the satellite images when it comes to sharpness and details but hardly when it comes to frequency and regularity. Satellite instruments record reflectance of electromagnetic waves of different wavelengths, primarily within the visual specter, blue, green and red color, but also infrared, and longer wave lengths. Reflectance of long waved radiation can be used to calculate temperature and humidity at the earth surface, terrestrial as well as aquatic [43]. Satellite surveillance can easily be done weekly, provided if the sky is not cloudy and the images are available on the Internet, many of them for free. By monitoring surface color, the concentration of chlorophyll a, suspended matter and Secchi depth can be estimated. If worrying values are observed, water samples may be taken to laboratory.

*Climate Change and Monitoring of Temperate Lakes DOI: http://dx.doi.org/10.5772/intechopen.84393* 

What topics, methods and experimental design should be recommended? During the last two to three decades, efforts are spent to reveal and predict effects of climate change, i.e., increase temperature and changed runoff patterns. Temperature trends alarmed scientists in the 1990s, and lake ecology was predicted to change if

#### **Figure 1.**

*Density of pelagic fish with body length* ≥ *approximately 5 cm recorded by means of a SIMRAD EY M echo sounder during 1986–2011 and by means of a SIMRAD EK 15 echo sounder in 2018 in Lake Osensjøen, Southeast Norway. Line shows moving average; vertical lines show 95% confidence intervals. For method description see Linløkken and Sandlund [40].* 

#### **Figure 2.**

*Length distribution of pelagic fish in four selected years (of Figure 1) based on echo target strength distribution from echo sounding in Lake Osensjøen, transformed to approximate fish lengths by the target strength—Fish length algorithm presented by Lindem and Sandlund [41].* 

this tendency continued [44]. Time series of temperature, combined with existing knowledge about temperature effects on fish ecology, affecting recruitment success and growth [45–48], differing between species, will benefit some species and opposite to others. The algae community reflects the lake's nutrition load but is also affected by the grazing zooplankton, which is affected by the zooplankton feeding fish [27]. Coregonids are important plankton feeders in cool and cold-water lakes, whereas cyprinids compete more efficiently as temperature and nutrition load increase and oxygen concentration decreases, and this is the most frequent in shallow lakes [8]. How can this be monitored and what changes can be observed so far?

 In the regulated and oligotrophic Lake Osensjøen in Southeast Norway, pelagic fish density, mainly whitefish and vendace, has been monitored by means of echo sounding since the 1980s, and during the first decade of this century, a pronounced density increase was observed, and a study was published in 2015 [40]. Year-class strength of both species was positively correlated with summer temperature, especially for vendace, as whitefish recruitment was also affected by the regulated water level (due to spawning sites at relatively shallow water). The vendace population increased, whereas whitefish in the pelagic zone decreased. A follow-up survey in 2018 confirmed high density of pelagic fish, and there was more than a sixfold increase since the 1980s and 1990s (**Figure 1**), and simultaneously, the proportion of fish >30 cm decreased, and the proportion of fish <20 cm increased (**Figure 2**). This has probably had an effect on the zooplankton community, of which samples are collected, but so far not analyzed. As the density increase was due to increased density of vendace, the growth rate of vendace has decreased [40].

#### **5. Monitoring of lakes using satellites**

Eutrophication and temperature increase may be expected to cause cyprinid dominance and amplify the effects of eutrophication through more intensive grazing on zooplankton. Temperature increase also affects recruitment and growth of percids positively [46, 48–50], among which perch and pikeperch are piscivorous and may play an important role in regulation of roach (*Rutilus rutilus*) and other cyprinids [51]. Exploitation with gill net fishing should be performed with great care to retain a sufficient number of large predatory specimens. What sufficient means should be a subject of research, which should otherwise concentrate on the description of species biomass of aquatic biotopes, with special attention on functional groups; who is eating who or what? Can it be stated an optimal balance between biomasses of consumers of first and second/third order, like between omnivorous/benhivorous/planktivorous cyprinids and species of higher trophic levels, like perch, pikeperch (*Sander lucioperca*) and pike and eventually the piscivorous cyprinid, the asp (*Aspius aspius*)? If not, serious measures may become necessary, like removal of fish biomass, which is shown to have a positive effect on zooplankton abundance, though not long lasting [52–54]. Stocking top predators, affecting the zooplanktivorous fish, may also have a positive effect on herbivorous zooplankton abundance [51, 55].

 To get an overview, lakes and river systems should be monitored by means of satellite images. These are available from several sources, and many are for free [43]. The resolution of the images varies, and whereas some satellites, like the Sentinel 3 A and B satellites, have rather low resolution with 300 × 300 m pixels in the visual wave specter, the two twin satellites pass every second day, that is, together they collect daily images of every spot of the inhabited world [56]. The Landsat 7 and 8 satellites depict an area every eighth day [43], and the Sentinel 2 A and B satellites do it every fifth day [57]. These pairs of satellites have pixels of 30 × 30 m and

#### *Climate Change and Monitoring of Temperate Lakes DOI: http://dx.doi.org/10.5772/intechopen.84393*

10 × 10 m, respectively, and the images are useful to characterize lakes with 5–10 km2 surface area. The images consist of different color bands, and these bands, and combinations of them, may be used to develop algorithms for environmental factors like chlorophyll a [43], if combined with in situ measurements. The distribution of colors nevertheless may give a clue of horizontal variation of, for example, chlorophyll.

In the southernmost East Norway, lakes of the Halden river system exhibits pronounced variation of trophic state. A satellite image of August 13, 2018, from Landsat 8, with manipulated pseudo colors showing the relationship between the reflectance of the green (reflected by chlorophyll) and the red (absorbed by chlorophyll) color bands, shows colors from blue to green, yellow, and orange/red (**Figure 3**). In the Lake Bjørklangen and Lake Hemnessjøen, the chlorophyll a concentration has through the years frequently been measured to 10–15 μg/l; in Lake Rødenessjøen it has been measured to 5–10 μg/l [58], whereas in the "blue" Lakes Setten and Rømsjøen [59], the chlorophyll a concentration is <5 μg/l. It must be added that the pixel values are also influenced by turbidity, that is, suspended solids, like in

#### **Figure 3.**

*Seven lakes in southernmost East Norway (Lat/Lon: 59°20′32″–59°56′60″, 11°02′ 13″–11°48′ 22″) showing horizontal variation of the ratio (pixel values of the green color band)/(pixel values of the red color band), based on a Landsat 8 satellite image, roughly indicating the distribution of chlorophyll [60].* 

the inlet of Lake Øyern and in the neighboring Glomma river system, which is not corrected for in this presentation.

In situ sampling, varying in space (horizontal) and time (seasonal), should be combined with satellite image analysis, to estimate reliable algorithms for the relationships between the chlorophyll a concentration, clarity and turbidity, and reflectance values (corrected pixel values) of different color bands of the satellite images. When this is established, satellite images can be used for regularly monitoring, daily in large lakes (>50 km2 ) and weekly in smaller lakes, though it will depend on the weather, that is, the cloudiness, which must be minor and surely not cover the lake. This can reveal point sources of pollution, and in situ sampling may be conducted during few days.

Fish monitoring is commonly done by gill net fishing or trawling, laborious and costly, and therefore is not conducted too often. As the pelagic fish stock is of special interest in eutrophication, hydro acoustic equipment is recommendable, and that can be done regularly, like every or every third or fifth year. The time of year and time of day affect the results, due to the spatial distribution of the fish, which must be in the pelagic zone and not too close to the surface to be recorded. An echo sounder counts the single fish, integrates schools to numbers of fish, and estimates density and target strength distribution which can be transformed to fish size distribution. This method can easily record increased density and changed size distribution, which is a probable result of climate change and is possibly the case in the Lake Osensjøen (**Figures 1** and **2**).

#### **6. Conclusion**

Lakes play important roles as ecological elements in nature and comprise important resources for human societies. They are also important as indicators of the ecological state of their catchment and are relatively easy to observe and monitor, by in situ sampling, by electronic logging, and by means of remote sensing from satellites. Thorough studies should be conducted to describe the at-present state of lakes, which is now largely taken care of through the EU Water Frame Directive, and it should be linked to monitoring routines by electronic logging devices and to remote sensing by means of satellite images. Algorithms relating reflectance to situ measurements should be worked out on a broad scale.

#### **Acknowledgements**

This chapter was supported by Hedmark University College/Inland Norway University College, and Glommen and Laagen Brukseierforening.

#### **Conflict of interest**

There is no conflict of interest.

*Climate Change and Monitoring of Temperate Lakes DOI: http://dx.doi.org/10.5772/intechopen.84393* 

### **Author details**

Arne N. Linløkken Inland Norway University of Applied Sciences, Hamar, Norway

\*Address all correspondence to: arne.linlokken@inn.no

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**53**

Section 4

Migration in Aquatic

Species

Section 4
