**7. Main methods used to evaluate cryopreserved semen**

#### **7.1. Sperm quality evaluation in fish**

Cryopreservation, although very useful from a production and conservation point of view, produces several types of damage to germ cells, especially to spermatozoa. Therefore, during the last decades huge efforts were done trying to reduce cryodamage, identify its causes and consequences to the sperm cell.

There are several constituents in a germ cell that can be analyzed. These analyses depend on the objective, possible applications, and on the methodology and available equipment. They can be from a simple motility analysis using a subjective scoring (normally used in fish farms) to more sophisticated analysis of motility, involving specific software (e.g., CASA system) or image analysis systems (Image J) [39]. Sperm quality can also be assessed by its constituents: seminal plasma and spermatozoa, indicating damage in specific cellular structures or in the entire cell, by releasing its constituents in the seminal plasma [39]. Determination of seminal plasma constituents and its variations such as lytic, oxidative, metabolic and apoptotic enzymes, metabolites, sugars, vitamins, amino acids, fatty acids, and other inorganic compounds can provide very useful information on sperm status [39]. Other characteristics of these cells should also be evaluated in order to assess sperm fertilization ability, especially when this assay is difficult to perform due to egg availability or long-term embryonic development. Most tests describing cell viability or mitochondrial status are currently performed with the use of fluorescent probes combined with microscopy or flow cytometry [40]. There is a huge list, depending on the main objective and available supplies and equipment, and can be from the determination of reactive oxygen species (ROS) levels, MDA concentration, detecting oxidative events at protein or lipid level, or simple cell structure impairments or ruptures. The assessment of sperm DNA is another test that should be performed since will provide information on sperm fitness being important for offspring quality. Methods to evaluate chromatin integrity include the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labeling), SCSA (sperm chromatin structure assay), and the analysis of specific DNA sequences using qPCR [41–43]. In recent years, and as an attempt to define bases for explaining other sperm quality markers at different levels, the characterization and presence of certain transcripts has been used [43]. These new tests can reveal the true meaning of spermatozoa quality.

All these techniques, although very useful in the evaluation of sperm quality and offspring viability, are still on a laboratory scale and need to be adapted for industry. Efforts are still required to transfer current technologies and make sperm banking accessible for fish producers [44]. In the last years, some national and international networks have been trying to get close contact between researchers and fish farm industries [45]. Although, there are still a lot of work to do, this topic should be considering as a priority step. It is also clear that although fundamental research is moving forward in the design of new methods and techniques to characterize sperm quality, it is important to focus on a process of scaling-up technologies to encourage companies and fish farmers in their use. Aquaculture needs to benefit from reproductive biotechnology.

#### **7.2. Quality evaluation in sperm from Brazilian species**

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

Cryopreservation, although very useful from a production and conservation point of view, produces several types of damage to germ cells, especially to spermatozoa. Therefore, during the last decades huge efforts were done trying to reduce cryodamage, identify its causes and

There are several constituents in a germ cell that can be analyzed. These analyses depend on the objective, possible applications, and on the methodology and available equipment. They can be from a simple motility analysis using a subjective scoring (normally used in fish farms) to more sophisticated analysis of motility, involving specific software (e.g., CASA system) or image analysis systems (Image J) [39]. Sperm quality can also be assessed by its constituents: seminal plasma and spermatozoa, indicating damage in specific cellular structures or in the entire cell, by releasing its constituents in the seminal plasma [39]. Determination of seminal plasma constituents and its variations such as lytic, oxidative, metabolic and apoptotic enzymes, metabolites, sugars, vitamins, amino acids, fatty acids, and other inorganic compounds can provide very useful information on sperm status [39]. Other characteristics of these cells should also be evaluated in order to assess sperm fertilization ability, especially when this assay is difficult to perform due to egg availability or long-term embryonic development. Most tests describing cell viability or mitochondrial status are currently performed with the use of fluorescent probes combined with microscopy or flow cytometry [40]. There is a huge list, depending on the main objective and available supplies and equipment, and can be from the determination of reactive oxygen species (ROS) levels, MDA concentration, detecting oxidative events at protein or lipid level, or simple cell structure impairments or ruptures. The assessment of sperm DNA is another test that should be performed since will provide information on sperm fitness being important for offspring quality. Methods to evaluate chromatin integrity include the comet assay (single cell gel electrophoresis), TUNEL (terminal deoxynucleotidyl transferase-nick-end-labeling), SCSA (sperm chromatin structure assay), and the analysis of specific DNA sequences using qPCR [41–43]. In recent years, and as an attempt to define bases for explaining other sperm quality markers at different levels, the characterization and presence of certain transcripts has been used [43]. These new tests can reveal the true meaning

All these techniques, although very useful in the evaluation of sperm quality and offspring viability, are still on a laboratory scale and need to be adapted for industry. Efforts are still required to transfer current technologies and make sperm banking accessible for fish producers [44]. In the last years, some national and international networks have been trying to get close contact between researchers and fish farm industries [45]. Although, there are still a lot

and optimizing the use of inseminating dose should be the goal for the near future.

**7. Main methods used to evaluate cryopreserved semen**

**7.1. Sperm quality evaluation in fish**

consequences to the sperm cell.

60 Cryopreservation in Eukaryotes

of spermatozoa quality.

According to Viveiros, Orfão [8] there are 18 Brazilian native species with high potential for cryopreservation, and indeed, several protocols have been developed and the postthaw quality assessed in most of those species (**Table 1**). Most of the work has been conducted in three main species, *P. lineatus, Brycon orbignyanus*, and *C. macropomum*. The first two more dedicated to conservation and slightly less to production and the *C. macropomum* more dedicated to production. In terms of postthaw quality analysis, research conducted in the last years, especially in these species gained high relevance and allowed to detect specific damage associated with cryopreservation. A subjective analysis of motility is still the main parameter analyzed, which in some cases can be responsible for the high variability of results obtained by different groups working in the same species [44]. The complementary analysis of other parameters such as cell viability using flow cytometer of fluorescent microscopy can help in this matter. Recent work in the previous mentioned species allowed to distinguish methyl glycol as the best cryoprotectant for *P. lineatus* and *B. orbignyanus* in terms of motility (63 and 72% motile cells, respectively) and cell viability (57 and 68% viable cells, respectively) [46]. A similar analysis concluded that DMSO and DMF (5 and 8%, respectively) were the best choices for freezing *C. macropomum* sperm, yielding motility and viability rates higher than 50%. Although, some damage was detected in postthaw samples, fertility, and hatching rates were not compromised (91.6 and 87.6%) [24, 47]. Therefore, it seems clear that although it is very important to perform a complete analysis of sperm using several indicators of quality such as motility, cell viability, and functionality and DNA status, fertility, and especially hatching rates are the ultimate tests to characterize the success of any cryopreservation protocol. These assays should be performed in order to demonstrate the viability of this technology to fish farmers, showing its applicability in aquaculture industry.


#### 62 Cryopreservation in Eukaryotes



**Species Assessed parameter Level of**

ultrastructure

Motility (CASA), viability, mitochondrial functionality

Motility (Subjective), fertilization

Motility (Subjective), fertilization rate, viability, mitochondrial functionality, DNA integrity

Motility (Subjective), fertilization rate, viability, mitochondrial functionality, DNA integrity

Motility (Subjective), fertilization rate, viability, mitochondrial functionality, DNA integrity

Motility (Subjective), fertilization

rate

*Leporinus macrocephalus* Motility (Subjective) Low Ribeiro and Godinho [69] *Leporinus obtusidens* Motility (Subjective) Low Viveiros, Maria [17]

*Piaractus brachypomus* Motility (Subjective-CASA) Low Nascimento, Maria [70]

rate, DNA methylation

*Brycon orthoenia* Motility (Subjective) Low Melo and Godinho [65]

rate

*Colossoma macropomum* Motility (Subjective), fertilization

*Brycon nattereri* Motility (Subjective),

62 Cryopreservation in Eukaryotes

**knowledge1**

motility (Subjective) Oliveira, Viveiros [60]

Motility (CASA) Viveiros, Gonçalves [62]

Motility, morphology (Subjective) Andrade, de Jesus [63] Motility, morphology (Subjective) Galo, Streit Jr [64] motility (Subjective) Viveiros, Maria [17]

Motility (Subjective) Carolsfeld, et al. [7]

motility (CASA) Leite, Oliveira [21] Motility (Subjective) Menezes, Queiroz [68]

motility (CASA) Melo-Maciel, Leite-Castro [67]

*Brycon opalinus* Motility (Subjective), viability Low Viveiros, Orfão [58] *Brycon orbignyanus* Motility (CASA) Medium López, Leal [61]

**References**

Viveiros, Nascimento [46]

Maria, Viveiros [16]

Garcia, Vasconcelos [20]

Varela Junior, Goularte [24]

Varela Junior, Corcini [47]

Taitson, Chami [26]

High de Mello, Garcia [66]

Low Viveiros, Maria [19]

1 Level of knowledge according to the used methodology on the postthaw sperm analysis of each species. Data have been collected from Web of Science platform since papers published from 2000 onwards by using as key words "species name + cryopreservation."

**Table 1.** Methods used for postthaw evaluation of sperm quality in freshwater Brazilian species.
