**2. Materials and methods**

#### **2.1. Reagents**

for transporting of genetic material between facilities, optimal using of gametes in aquaculture, reducing risk of spreading infections, performing of hybridization studies, conserving of

Cryopreservation technique involves addition of cryoprotectants to the extender and freezing and thawing of sperm samples, which may result in some damage to the spermatozoa and may decrease egg fertilization rate. Therefore, before cryopreservation of spermatozoa, a thorough evaluation of different extender solutions, cryoprotectants, and cooling and thawing rates are essential to develop optimum cryopreservation protocol for various species [3–5].

During the cryopreservation process, some factors may change the physiological status of sperm. The success of cryopreservation depends not only on preserving the motility of the spermatozoa but also on maintaining their metabolic functions [6]. Extender composition and cryoprotectant concentration are the main factors affecting cryopreservation success [7]. Extenders are required for dilution of fish sperm prior to cryopreservation and are generally designed to be compatible with the physiochemical composition of the fish seminal plasma. Most important function of the extenders is to maintain the spermatozoa in immotile state until

Cryoprotectants are added to the extenders to protect the cells against ice crystal formation during freezing and thawing [9]. Although cryoprotectants help to the prevention of cryoinjuries during freezing and thawing, they may become toxic to the cells when exposure time and concentration are increased [10, 11]. Thus, one of the most critical steps in successful cryopreservation of fish semen is the choice of the cryoprotectant and its ratio in the extender

Another important problem is the handling of sperm produced in small volumes by some fish species such as tilapia. In spite of packaging of sperm in traditional 0.25-mL and 0.5-mL straws has been successfully applied to freeze semen of the most fish species and to fertilize small egg batches [9], there is a lack of information regarding their usage in cryopreservation of Nile

The Nile tilapia is one of the most cultivated freshwater fish species in the world aquaculture [12]. This species has great breeding potential due to its hardiness against worse environmental conditions, fast growth rate, adaptation to different environmental conditions (e.g. salinity, temperature), and also good organoleptic characteristics of its flesh [13, 14]. On the other hand, most of studies related with fish sperm cryopreservation have focused on some freshwater species, such as cyprinids [15, 16], salmonids [17, 18], catfishes [19, 20], and loach [21].

Even though many successes have been achieved in fish semen cryopreservation, the technique remains as a method that is difficult to standardize and use in all types of fishes. This is due to the fact that cryopreservation of sperm from different fish species required different conditions, where the protocol needs to be established individually [22]. To the best of our knowledge, there is limited information regarding cryopreservation of Nile tilapia sperm. In this concept, the effect of cryoprotectants and packaging methods on freezability and also on post-thaw quality of Nile tilapia sperm still remains unclear. Thus, standardization and

protecting endangered species, and also for conserving of biodiversity [1, 2].

required [8].

76 Cryopreservation in Eukaryotes

during the process.

tilapia (*Oreochromis niloticus*) semen.

The additives and other chemicals used in this study were obtained from local representative of Sigma-Aldrich Chemicals Company (St. Louis, MO, USA).

#### **2.2. Broodstock handling**

The experiments were carried out spawning season of the Nile tilapia. In the pre-spawning period, sexually mature male (n=15) and female (n=5) Nile tilapia were pit-tagged and kept separately in 150 L indoor tanks under constant environmental conditions. The broodstock tanks were provided with freshwater constantly at ratio of 1.5 L/s, while compressed air was provided trough air stones. The water temperature ranged from 27.2°C to 30.5°C, and salinity was maintained at 1.5 ppm. Nile tilapia was fed with floating pellets twice daily (1–5% body weight per day).

#### **2.3. Gamete collection**

Gametes were collected from healthy mature males and females following immersion anesthetization with 10 ppm quinaldine (Reanal Ltd., Budapest, Hungary) for a few minutes. For sperm collection, 1-mL tuberculin plastic syringe, without needle, was used to aspirate sperm released by gentle abdominal massage to eliminate urine in the ducts. Following, sperm samples were transferred individually into 1.5-mL Eppendorf tubes on ice (0–4°C). A 10-μL pipette tip connected to a mouth pipette was used to extract sperm cells, which were diluted 1:1 in Hanks' balanced salt solution (HBSS) (280 mOsm/kg, pH 7.0) in 1.5-mL microcentrifuge tubes and placed on ice until analysis. Eggs were also collected by gentle abdominal massaging and stored in HBSS at 25°C and used for fertilization within 30 min following stripping [23].

#### **2.4. Gamete quality determination**

One microlitre sperm of each sample was placed on a microscope slide and observed under a phase-contrast microscope (Olympus, Japan) at 100× magnification. The motility characteristics of the collected sperm samples were evaluated by adding activation solution (AS) (45 mM NaCl, 5 mM KCl, and 30 mM Tris–HCl, pH 8.2) [24] at a ratio of 1:100. Sperm cells that vibrated in place were considered as immotile. Only samples whose quality parameters ranging between the following values were used for the cryopreservation experiment: osmolarity 50– 100 mOsm/kg, pH 7.0–8.0, and progressive motility 80–100% [25]. The quality of ova was determined from their morphological features seen under a dissecting microscope as described in the study of Fauvel et al. [26].

#### **2.5. Cryopreservation and thawing experiments**

Semen samples showing ≥80 motility was pooled into equal aliquots and chosen for cryopreservation experiments. Semen and extenders were kept at 4°C immediately under aerobic conditions prior to dilution. Pooled semen was diluted at a ratio of 1:3 with an extender composing 350 mM glucose and 30 mM Tris [27] containing 10% dimethylacetamide (DMA). The diluted semen were drawn into 0.25-mL or 0.5-mL plastic straws (IMV France) and sealed with polyvinyl alcohol (PVA). Following equilibration of semen for 10 min at 4°C, the straws were placed on a styrofoam rack floating on the surface of liquid nitrogen in a styrofoam box. Samples were frozen 3 cm above of the liquid nitrogen surface and exposed to the liquid nitrogen vapor (≈−140°C) for 10 min [28]. Following, frozen sperm cells were kept into the liquid nitrogen container (−196°C) until analyses for a few days.

The frozen sperm in different volume of straws were thawed in a water bath at 30°C for 20 s (0.25-mL straws) or at 30°C for 30 s (0.5-mL straws). Thawed semen was activated using activation solution (AS) (45 mM NaCl, 5 mM KCl, and 30 mM Tris–HCl, pH 8.2) [24] and observed under a phase-contrast microscope (Olympus, Japan) for progressive motility (%), progressive motility duration (s), and viability (%) evaluations (three replicates). At least five straws were used for each parameter evaluation with three replications.

#### **2.6. Post-thaw sperm quality determination**

The percent of motile spermatozoa and motility duration was immediately recorded following activation using a CCD video camera (CMEX-5, Netherland) mounted on a phase-contrast microscope (100×, Olympus BX43, Tokyo, Japan) at room temperature (20°C). Progressive spermatozoa motility and duration of progressive spermatozoa motility were evaluated from sperm with forward movement. Immotile spermatozoa were defined as spermatozoa that did not show forward movement after activation. Percentage of spermatozoa motility was determined within 30 s post-activation. Motility duration was evaluated by counting the time from spermatozoa activation until spermatozoa stopped moving. In order to assess viable sperm percentage, eosin-nigrosin preparations were made according to the method described by Bjorndahl et al. [29] and totally 300 sperm cells were counted on each slide at 1000× magnification. At least five straws were used for each evaluation parameter, and analyses were repeated three times for each treatment.

#### **2.7. Fertilization experiments**

Pooled eggs from five mature females were used to assess fertilization rates. In this stage, most of the HBSS was decanted from the eggs, and fertilization was carried out in dry Petri dishes (10 cm diameter). Fresh or thawed sperm was added over the eggs and gently mixed before activation with 20 mL of fertilization solution (3 g urea and 4 g NaCl in 1 L distilled water) [30]. Following fertilization process, 2 mL embryo buffer medium (EBM) (13.7 mM NaCl, 5.40 mM KCl, 0.25 mM Na2HPO4, 0.44 mM KH2PO4, 1.30 mM CaCl2, 1.00 mM MgSO4, 4.20 mM NaH-CO3 at 52 mOsm/kg and pH 7.0) was added for activation as described by Westerfield [31]. After 10 min, 100 mL EBM was added again over the eggs and was left undisturbed without movement in a convection type incubator at 27°C (Panasonic MCO-19M-PE, Japan). Unfertilized eggs were removed, and EBM was changed twice daily. After 48 h, the eggs were evaluated for fertilization results. Eggs that developed to stage 11 (embryonic keel and somite formation) were recorded as fertilized eggs, described by Galman and Avtalion [32].

Fertilization experiments were carried out using 1×105 :1 spermatozoa/egg ratio with each straw types (0.25 or 0.5 mL) for the each aliquot of eggs (containing 100 eggs). Three straws were thawed for each fertilization treatment (three replications). For the control, another three aliquot of eggs (containing 100 eggs) were fertilized with fresh semen collected from two other males. Eggs were fertilized with fresh semen samples using the same number of sperm cells (1 × 105 cells) similar to treatments with frozen semen.

#### **2.8. Statistical analysis**

A two-way analysis of variance (ANOVA) including the straw volumes (0.25 and 0.5 mL) as fixed effects was used. Means were separated by Duncan's multiple range test and were considered at 5% level of significance. Results are presented as mean ± S.D. All analyses were carried out using SPSS 17 for Windows statistical software package.

## **3. Results**

between the following values were used for the cryopreservation experiment: osmolarity 50– 100 mOsm/kg, pH 7.0–8.0, and progressive motility 80–100% [25]. The quality of ova was determined from their morphological features seen under a dissecting microscope as described

Semen samples showing ≥80 motility was pooled into equal aliquots and chosen for cryopreservation experiments. Semen and extenders were kept at 4°C immediately under aerobic conditions prior to dilution. Pooled semen was diluted at a ratio of 1:3 with an extender composing 350 mM glucose and 30 mM Tris [27] containing 10% dimethylacetamide (DMA). The diluted semen were drawn into 0.25-mL or 0.5-mL plastic straws (IMV France) and sealed with polyvinyl alcohol (PVA). Following equilibration of semen for 10 min at 4°C, the straws were placed on a styrofoam rack floating on the surface of liquid nitrogen in a styrofoam box. Samples were frozen 3 cm above of the liquid nitrogen surface and exposed to the liquid nitrogen vapor (≈−140°C) for 10 min [28]. Following, frozen sperm cells were kept into the

The frozen sperm in different volume of straws were thawed in a water bath at 30°C for 20 s (0.25-mL straws) or at 30°C for 30 s (0.5-mL straws). Thawed semen was activated using activation solution (AS) (45 mM NaCl, 5 mM KCl, and 30 mM Tris–HCl, pH 8.2) [24] and observed under a phase-contrast microscope (Olympus, Japan) for progressive motility (%), progressive motility duration (s), and viability (%) evaluations (three replicates). At least five

The percent of motile spermatozoa and motility duration was immediately recorded following activation using a CCD video camera (CMEX-5, Netherland) mounted on a phase-contrast microscope (100×, Olympus BX43, Tokyo, Japan) at room temperature (20°C). Progressive spermatozoa motility and duration of progressive spermatozoa motility were evaluated from sperm with forward movement. Immotile spermatozoa were defined as spermatozoa that did not show forward movement after activation. Percentage of spermatozoa motility was determined within 30 s post-activation. Motility duration was evaluated by counting the time from spermatozoa activation until spermatozoa stopped moving. In order to assess viable sperm percentage, eosin-nigrosin preparations were made according to the method described by Bjorndahl et al. [29] and totally 300 sperm cells were counted on each slide at 1000× magnification. At least five straws were used for each evaluation parameter, and analyses were

Pooled eggs from five mature females were used to assess fertilization rates. In this stage, most of the HBSS was decanted from the eggs, and fertilization was carried out in dry Petri dishes (10 cm diameter). Fresh or thawed sperm was added over the eggs and gently mixed before

in the study of Fauvel et al. [26].

78 Cryopreservation in Eukaryotes

**2.5. Cryopreservation and thawing experiments**

**2.6. Post-thaw sperm quality determination**

repeated three times for each treatment.

**2.7. Fertilization experiments**

liquid nitrogen container (−196°C) until analyses for a few days.

straws were used for each parameter evaluation with three replications.

Semen volume was rather variable and ranged from 0.9 to 7.5 mL, with a mean volume of 3.6 ± 0.40 mL. Progressive motility was ranged from 60% to 90%, and mean motility was determined as 80.4 ± 0.15%. In addition, mean progressive spermatozoa motility duration (s),

**Figure 1.** Post-thaw progressive motility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).

spermatozoa density (× 109 /mL), viability (%), and pH values were determined as 64.2 ± 0.45 s, 1.75 × 109 /mL, 92.5 ± 4.25%, and 7.2 ± 0.25, respectively. In addition, mean fertilization rate was determined with fresh semen, which was 72.5 ± 0.20%. The findings of the present study indicated that cryopreservation of sperm in glucose-Tris–based extender using 0.5-mL straws increased post-thaw progressive motility (**Figure 1**), duration of progressive motility (**Figure 2**), and fertility (**Figure 4**) (P<0.01). On the other hand, differences in terms of postthaw cell viability were not significant among the treatments (**Figure 3**, P>0.01). The fertility of the frozen-thawed sperm showed high positive linear correlation with motility (r2 =1.000, **Figure 5**) and (r2 =0.9932, **Figure 6**) in case of using 0.25-mL and 0.5-mL straws.

**Figure 2.** Post-thaw progressive motility duration (s) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).

**Figure 3.** Post-thaw viability (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).

spermatozoa density (× 109

80 Cryopreservation in Eukaryotes

s, 1.75 × 109

**Figure 5**) and (r2

/mL), viability (%), and pH values were determined as 64.2 ± 0.45

=1.000,

/mL, 92.5 ± 4.25%, and 7.2 ± 0.25, respectively. In addition, mean fertilization rate

was determined with fresh semen, which was 72.5 ± 0.20%. The findings of the present study indicated that cryopreservation of sperm in glucose-Tris–based extender using 0.5-mL straws increased post-thaw progressive motility (**Figure 1**), duration of progressive motility (**Figure 2**), and fertility (**Figure 4**) (P<0.01). On the other hand, differences in terms of postthaw cell viability were not significant among the treatments (**Figure 3**, P>0.01). The fertility of the frozen-thawed sperm showed high positive linear correlation with motility (r2

=0.9932, **Figure 6**) in case of using 0.25-mL and 0.5-mL straws.

**Figure 2.** Post-thaw progressive motility duration (s) of Nile tilapia sperm cryopreserved with glucose-Tris–based ex-

**Figure 3.** Post-thaw viability (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns

tender. Columns marked with different letters are significantly different (P<0.01, n=3).

marked with different letters are significantly different (P<0.01, n=3).

**Figure 4.** Post-thaw fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender. Columns marked with different letters are significantly different (P<0.01, n=3).

**Figure 5.** Relationship between post-thaw spermatozoa motility (%) and fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender using 0.25-mL straws.

**Figure 6.** Relationship between post-thaw spermatozoa motility (%) and fertility (%) of Nile tilapia sperm cryopreserved with glucose-Tris–based extender using 0.50-mL straws.
