**6. Artificial fertilization**

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

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

Freezing and thawing rates are determining factors in a gamete cryopreservation protocol. A standard set for the protocols adopted for the South American native fish species is something lacking. In the published studies, we can observe different freezing and thawing rates for different species and even for the same species. Surely, this implies difficulty of adopting a technology that could be replicated and used efficiently by a farmer or even a trained technician. The use of a dry shipper has been established as a common method in research with semen cryopreservation in Brazil [8], since is considered a safe and practical method [24]. Moreover, the dry-shipper container used in research produces similar freezing rates and storage temperatures [13, 25, 26]. However, it is in the thawing rates that the largest variations are noticed, since different temperatures and thawing times are used [8]. For example, Velasco-Santamaría et al. [27] tested 0.5, 1.8, 2.5, and 4.0 ml straws for freezing of *Brycon amazonicus* semen and the results indicated that an increased in thawing temperature from 35 to 80°C for 10 or 90 s could influence sperm motility and fertilization rate. However, for *P. lineatus* the thawing temperature may vary from 30 to 60°C for 8 or 16 s, respectively, without major changes in motility rate when using 0.5 ml straws [18]. For *C. macropomum* semen, a thawing temperature of 45°C for 5–8 s [20, 24] when using 0.25 ml straw and for 0.5 ml straw 37°C/30 s or 60°C/8 s was used [13, 21]. However, at larger scale using 1.6 or 4.5 ml straws these rates

Note that the size of the straw can affect the quality of cryopreserved semen [28] as well as the temperature used for thawing, since high temperatures can denature enzymes and proteins of sperm cells [29]. Therefore, it is necessary to search for a balance between thawing rate and especially size of the container, to be able to maximize the use of cryopreserved semen and

By having a limited supply of ATP accumulated in the mitochondria [30], the motility duration in freshwater fish spermatozoa is greatly reduced. Motility is a variable that significantly alter the sperm's ability to fertilize the oocyte [31]. Therefore, the use of balanced osmotic media

parts of the country.

58 Cryopreservation in Eukaryotes

species should be searched.

can be of 60°C for 90 s [25].

**5. Activating solutions**

have a product in quality and quantity [25].

**4. Freezing and thawing rates**

Most papers on seminal cryopreservation from South American fish do not have the data of fertilization rates using cryopreserved semen [8]. The final validation of applying this technique necessarily culminates with the results of fertilization and hatching rates. Nevertheless, to reach this goal some steps must comply in order to create a favorable environment allowing the encounter of the gametes to occur in an efficient and safe manner. In Brazil, in general the most basic aspects involving fish gametes manipulation are still neglected on fish farms. There are preeminent need to establish guidelines for best practices in fish handling during gametes collection and all subsequent semen handling until freezing.

Generally, fertilization rates are lower when using cryopreserved semen, which is a function of reduced quality of sperm cells. Regarding the lack of studies assessing the fertilization and hatching rates, we can relate: the difficulty of having oocytes available for carrying out the tests considering the maintenance costs of a broodstock; experiments poorly designed and lack of methodology standardization in the studies. It is noteworthy that even a motile sperm is no guarantee of fertilization. From more elaborated analysis, discussed in the next topic, it is possible to check for sperm membrane or DNA damage and how such damage can affect their fertilizing capacity.

Indeed, for development of a commercial protocol using cryopreserved semen in Brazil, even on a small scale, an evolution in the control of the above-mentioned processes will be required. If the ultimate goal is to get a good hatching rate (live larvae), that is directly related to the quality of biological material to be cryopreserved, asepsis of facilities and equipment and an efficient cryopreservation protocol. We must consider that the quality of oocytes should be excellent, ensuring that this is not a factor that negatively interfere to the success of the procedure. Are there any criteria for assessing the spawning quality of fish species studied in Brazil? The answer is no, there are not! Nowadays, there is no effective technique to evaluate the quality of oocytes in fish farming in a practical and affordable way.

The inseminating dose (spermatozoa/oocyte ratio) is an important feature that starts to be focused in some studies [36–38]. The entire control of the artificial fertilization environment and optimizing the use of inseminating dose should be the goal for the near future.
