**3. Cryoprotectants**

glucose as nonpermeating cryoprotectant. Other fish species have been studied at that period,

The 1990s began with the studies focused on the species target as potential for production in the fish farming scenario at that time, the *Piaractus mesopotamicus* [4, 5]. However, during this decade, there were virtually no further researches in the area and only in 1999 a study with *Colossoma macropomum* [6] was published. However, the study considered the major milestone in fish semen cryopreservation in Brazil was carried out by Carolsfeld et al. [7], who published a series of compiled results describing the freezing protocols for native species, most of them still being used nowadays. Currently, around 20 Brazilian freshwater species have their semen cryopreserved, some even have different protocols [8], or small variations from the protocols

The studies that guided the composition of a future protocol for fish semen freezing produced a lack of originality, resulting in little progress to date. Some factors have contributed to this scenario, such as: the small number of researchers working in the area; restriction of modern equipment; reduced scientific exchange with international groups and even the lack of criteria for distribution of public resources that result in numerous similar publications without

This chapter is not intended to point out the successes and mistakes that may have been committed in the pursuit of development of cryopreservation protocols for fish semen in Brazil, but a reflection on the future directions considering what should be pondered on this subject

The extenders are used to meet several requirements necessary for sperm survival during cryostorage. Its function is to provide a favorable microenvironment to maintain the viability of sperm cells, allowing the addition of energy sources (lipids, carbohydrates, and metabolites) to support the cellular metabolism, control of pH and osmolality, and prevention of bacterial

Since many factors can influence on semen quality parameters, the extender solution must be carefully formulated [10]. The first extender mediums used for South American fish semen were composed only by 0.8% NaCl [1]. Thereafter, an improvement in the results was noticed

Egg yolk is still frequently used as a component of extender solutions. According to Watson [11], due to the low-density lipoprotein (LDL) fraction in its composition, the egg yolk may provide a better stabilization of the sperm membrane by passing the cryopreservation stress, reducing injuries, and the thermal shock. The stabilizing mechanism of the sperm membrane by LDL possibly occurs because these compounds attract the molecules of cholesterol present in seminal plasma, thus preventing cholesterol binds to the phospholipids of the sperm

when glucose was added to the mediums, jointly with egg yolk or powder milk [7].

such as *Rhamdia hilarii = R. quelen* [2] and *Leporinus silvestrii* [3].

described by Carolsfeld et al. [7].

with objectivity and scientific consolidation.

significant progress.

56 Cryopreservation in Eukaryotes

**2. Extender solutions**

growth [9].

For performing cryopreservation, either by slow freezing or ultrarapid techniques, the use of permeating and nonpermeating cryoprotectant agents (CPAs) is required [22]. However, CPAs can be very toxic depending on the concentration used and cell exposure to them before freezing should be controlled [22].

The nonpermeable CPA most used by Brazilian researchers for semen from scale fish species is dimethylsulfoxide (DMSO) [8], whereas for nonscale fish species methanol is the most commonly used CPA [7, 23]. In the last years other CPAs have also been tested, such as methyl glycol, glycerol, ethylene glycol, and dimethylformamide (DMF) [8]. We understand that testing new CPAs should be encouraged in research. However, it is important to consider some aspects and thus avoid wasting of time and money. DMSO is no doubt the CPA that has shown the best results to date. We must understand very well the chemical and physical behaviors of a given reagent at low temperatures as well as its permeability and toxicity to sperm cells to be worth DMSO's replacement.

Glucose, as a low molecular weight nonpermeable CPA has been widely used in most protocols in Brazil. However, it is still quite common to observe its use for the formulation of cryoprotectant solutions with egg yolk [8]. Among the cryoprotective effects of glucose on sperm cells are dehydration before cooling, leading to less intracellular ice crystal formation, the increase of effective viscosity of the media, and serving as a protector to membrane integrity. Importantly, glucose is a component commonly added to the cryoprotectant solution and its function as an energy supplier to the spermatozoon and/or cryoprotective action will depend on the concentration used.

We consider as a decisive factor for the composition of the cryomedium not only the choice of cryoprotectants, but also their chemical quality. Although it appears to be negligible, this fact is crucial for a proper and successful protocol. There has been a significant improvement in the supply of chemical reagents, but the delivery time and costs remain a limiting in some parts of the country.

The large number of farmed fish species nowadays in Brazil may be one of the causes of using a high range of different CPAs. However, a standardization of cryoprotocols within a given species should be searched.
