**2. Extender solutions**

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 growth [9].

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

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 membrane, avoiding their destabilization [12]. In *C. macropomum*, the addition of egg yolk in the extender improved sperm motility rate [13].

The composition of the extenders used for preservation of semen from South American fish is quite varied and may be based on salts, glucose, or a bit more complex composition, which includes the addition of energy substrates and antibiotics [14, 15].

The extenders used for the South American fish species are still based on solutions formulated for mammal's semen. Currently, the BTS™ (Beltsville Thawing Solution—Minitub), which is formulated for swine semen is the basic solution that has been used in the composition of extenders [16–19]. Similarly, ACP-104™ (ACP Biotechnology—UECE), which was originally formulated for goats semen was also tested in some fish species [15, 17, 20, 21]. However, seminal viability results obtained in the literature are very variable, indicating that there is a need to seek efforts to formulate specific extenders, based on biochemical composition of seminal plasma from each fish species. Knowledge on the biochemical characteristics of the seminal plasma is essential to understand the spermatozoa requirements, assisting in the preparation of appropriate extender solutions for both short and long-term cryostorage.
