**4. Freezing and thawing rates**

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 can be of 60°C for 90 s [25].

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 have a product in quality and quantity [25].
