**4. Discussion**

Tilapias are widely cultured in the tropical and subtropical regions of the world. Several species of tilapia are cultured commercially, but Nile tilapia is the predominant cultured species worldwide that its production reached 3,197,330 mt in 2012 [33].

Although Nile tilapia is a freshwater fish, it can tolerate a wide range of salinity [34]. Therefore, the expansion of its culture in sea and brackishwater has attracted the attention of fish farmers in recent years. However, limited reports have addressed semen cryopreservation in the Nile tilapia. Therefore, standardization and simplification of the cryopreservation procedure for the Nile tilapia sperm is needed for commercial application. On the other hand, because of limited amounts of data are available, comparison of the methods and results have been mainly made with the cyprinid species in this research. From this point of view, findings of this research significantly contribute improving of the protocols applied for the cryopreservation of the Nile tilapia sperm.

Sperm cryopreservation is an important biotechnological technique with specific advantages to the aquaculture industry. Improvements in semen cryopreservation techniques require indepth knowledge of gamete physiology and the biochemical processes occurring during semen collection, processing, freezing, and thawing [35].

In spite of routinely using of cryopreserved semen in aquaculture artificial insemination programs worldwide, there are inconsistencies in experimental results [36]. The success of cryopreservation mainly depends on maintaining the spermatozoal metabolic functions [37]. The major factors affecting the results of insemination with frozen/thawed sperm are the type and properties of extenders and cryoprotectants, the damage caused by the formation of internal ice crystals due to the increase in solute concentration in the extension media and the interaction of these factors [38].

In the present study, glucose-Tris–based extender supplemented with DMA was used to cryopreserve Nile tilapia sperm. In spite of using glucose mainly as energetic substrate, it has been used due to its stabilization effects on the spermatozoa liposomal membrane [39]. It should be noted that, carbohydrate-based solutions such as glucose have also been found effective in some experiments [40, 41], and Tris is the most common buffer solution, not only for cyprinidae but also for other fish species [42].

Cryoprotectants are added to the extenders in order to protect the spermatozoa from damages during the freezing/thawing process. On the other hand, cryoprotectants can suppress most cryoinjuries when used at higher concentrations but at the same time it can become toxic to the cells. In addition, the amount and type of cryoprotectants used in sperm diluent depend on fish species and can affect the physiological and metabolic structure of the spermatozoa during cryopreservation procedure in different ways [43]. Therefore, selection of suitable type and concentration of the cryoprotectant is needed for the development of an effective cryopreservation protocol. However, comparison of different cryoprotectants and freezing/ thawing protocols are difficult when each treatment tested for the ability of fertilization of the eggs by spermatozoa. For this reason, the protective effect of different cryoprotectants varies

in different fish species. In this concept, several cryoprotectants have been mainly used for fish sperm cryopreservation, such as dimethyl sulphoxide (DMSO), dimethyl acetamide (DMA), glycerol (Gly), methanol (MeOH), ethylene glycol (EG), and propylene glycol (PG) [44–46].

**4. Discussion**

82 Cryopreservation in Eukaryotes

of the Nile tilapia sperm.

interaction of these factors [38].

Tilapias are widely cultured in the tropical and subtropical regions of the world. Several species of tilapia are cultured commercially, but Nile tilapia is the predominant cultured species

Although Nile tilapia is a freshwater fish, it can tolerate a wide range of salinity [34]. Therefore, the expansion of its culture in sea and brackishwater has attracted the attention of fish farmers in recent years. However, limited reports have addressed semen cryopreservation in the Nile tilapia. Therefore, standardization and simplification of the cryopreservation procedure for the Nile tilapia sperm is needed for commercial application. On the other hand, because of limited amounts of data are available, comparison of the methods and results have been mainly made with the cyprinid species in this research. From this point of view, findings of this research significantly contribute improving of the protocols applied for the cryopreservation

Sperm cryopreservation is an important biotechnological technique with specific advantages to the aquaculture industry. Improvements in semen cryopreservation techniques require indepth knowledge of gamete physiology and the biochemical processes occurring during

In spite of routinely using of cryopreserved semen in aquaculture artificial insemination programs worldwide, there are inconsistencies in experimental results [36]. The success of cryopreservation mainly depends on maintaining the spermatozoal metabolic functions [37]. The major factors affecting the results of insemination with frozen/thawed sperm are the type and properties of extenders and cryoprotectants, the damage caused by the formation of internal ice crystals due to the increase in solute concentration in the extension media and the

In the present study, glucose-Tris–based extender supplemented with DMA was used to cryopreserve Nile tilapia sperm. In spite of using glucose mainly as energetic substrate, it has been used due to its stabilization effects on the spermatozoa liposomal membrane [39]. It should be noted that, carbohydrate-based solutions such as glucose have also been found effective in some experiments [40, 41], and Tris is the most common buffer solution, not only

Cryoprotectants are added to the extenders in order to protect the spermatozoa from damages during the freezing/thawing process. On the other hand, cryoprotectants can suppress most cryoinjuries when used at higher concentrations but at the same time it can become toxic to the cells. In addition, the amount and type of cryoprotectants used in sperm diluent depend on fish species and can affect the physiological and metabolic structure of the spermatozoa during cryopreservation procedure in different ways [43]. Therefore, selection of suitable type and concentration of the cryoprotectant is needed for the development of an effective cryopreservation protocol. However, comparison of different cryoprotectants and freezing/ thawing protocols are difficult when each treatment tested for the ability of fertilization of the eggs by spermatozoa. For this reason, the protective effect of different cryoprotectants varies

worldwide that its production reached 3,197,330 mt in 2012 [33].

semen collection, processing, freezing, and thawing [35].

for cyprinidae but also for other fish species [42].

Cryoprotectants are essential for preservation, but it is dissimilarly toxic to the cells. The toxicity tolerance level of the cells also depends on cryoprotectant concentration. Also, there are differences in permeability of the cells according to cryoprotectant types. In this study, DMA as penetrating cryoprotectant was used at 10% concentration, and diluted samples were equilibrated for 10 min at 4°C. Some authors recommend having an equilibration period following dilution, allowing cryoprotectants to penetrate the spermatozoa before cryopreservation [9, 47]. However, some authors reported equilibration process did not improve cryopreservation success in fish [48, 49].

On the other hand, the freezing conditions depended on the straw size and were also species specific. Insemination with cryopreserved semen of Arctic charr (*Salvelinus alpinus*) in 1.7-mL flat straws using 10% DMSO resulted highest percentage of eyed eggs (57.9 ± 11.6%) than with 0.5-mL and 2.5-mL straws [50]. No significant difference was obtained between fertilization percentage of blue catfish spermatozoa frozen in 0.5-mL and 1-mL straws. The larval hatch rates of striped trumpeter (*Latris lineata*) semen frozen in 0.25-mL and 0.5-mL straws (44.3 ± 2.9% and 44.2 ± 2.0%) [51]. In case of common carp, the use of conventional 0.5-mL straws resulted in 67 ± 17% hatching [24]. The findings of the present study demonstrated that progressive motility decreased for the two types of tested straws and varied from 55.7% in 0.5 mL to 40.2% in 0.25-mL straws. In addition, the 0.5-mL straws gave the best results in fertility as 45.7% when compared with 0.25-mL straws. The use of these small volumed straws during the artificial reproduction in fish can be time consuming, as many straws are needed to fertilize thousands of eggs. On the other hand, small volumed straws are more suitable for gene banking or fertilization of small amounts of eggs under laboratory conditions.

Successful fertilization of eggs using cryopreserved sperm is the final target of cryopreservation process. Fertilization ability of the cryopreserved sperm is a reliable approach to evaluate success of the cryopreservation protocol [52]. According to the results of the present study, Nile tilapia spermatozoa were influenced by cryopreservation process, and depending on this interaction, fertilization ability of frozen/thawed sperm decreased than fresh ones. The reason for the low fertility rate of frozen/thawed spermatozoa may be attributed to the changes in ultrastructural morphology, decrease in progressive motility and motility duration, and also possible toxic effects of the DMA.

Motility is one of the most important factors to assess fish sperm quality because it gives important information about the sperm cell's energy sources. In addition, better knowledge of the characteristics of fresh sperm motility is necessary to evaluate sperm quality in commercial hatcheries before artificial reproduction and also in laboratories before experiments. Spermatozoa motility is induced following releasing of the spermatozoa into the aquatic environment during natural reproduction or after transferring to an activation medium during controlled reproduction [53].

The observed decrease in sperm motility might be due to decrease in the percentage of sperm viability, high damage of sperm cells, or decrease in ATP content following cryopreservation. Similarly, Alavi et al. [54] determined that almost all studies on sturgeon sperm cryopreservation showed significant lower sperm motility and fertilizing ability of frozen/thawed sperm compared to that of the fresh sperm.

On the other hand, when fish spermatozoa are released into water or activation medium, they have a brief spermatozoal activity period [55]. For instance, in fresh sperm, the duration of spermatozoa motility in several cyprinids have been reported to last 120 s [56]. Similarly, in case of silver barb, the maximum motility period was observed until 150 s after water activation [4]. However, in case of frozen/thawed sperm, duration of mean post-thaw spermotozoa motility (32.0 ± 8.16 s) of the Nile tilapia was determined as lower than the results assessed with mirror carp [57] but higher than that of scaly carp [46] when DMSO, DMA, and glycerol were used as cryoprotectant. Similar results for the motility parameters of frozen-thawed spermatozoa were reported in fish in some experiments [48, 58, 59]. On the other hand, it is interesting to note that Godinho et al. [60] reported 241.2 ± 57.3 s post-thaw spermatozoa motility duration in glucose-based cryosolution containing 10% methanol in Nile tilapia.

In the present study, the applied sperm/egg ratio was 1×105 :1 for fresh as well as frozen/thawed sperm, which probably resulted in excessive sperm concentrations in all batches. However, according to Lubzens et al. [48], the concentration of frozen/thawed sperm to be used to achieve optimal fertilization and hatching success is approximately 100 times higher than for fresh semen. This may be due to differences in extender compositions, cryoprotectant types, equilibration periods, egg quality, or applied protocols. In the present study, high positive correlation was determined between post-thaw spermatozoa motility and fertilization. This situation was consistent with the results that obtained from turbot (*Psetta maxima*) [61], common carp (*Cyprinus carpio*) [49], and African catfish (*Clarias gariepinus*) [62].
