**3. Results**

296 Current Frontiers in Cryopreservation

compression during freezing and thawing (Lahnsteiner, 2000). Following sperm suspension

Within 1 h after sperm collection, the diluted semen samples were drawn into 0.25mL plastic straws (IMV, France). The open end of straws were sealed with polyvinyl alcohol (PVA). Following, the straws were placed on a styrofoam rack that floating on the surface of liquid nitrogen in a styrofoam box. The straws were frozen in liquid nitrogen vapour 4 cm above of the liquid nitrogen surface (temperature of styroframe surface was about -140ºC) for 10 min. Following, the straws were plunged into the liquid nitrogen (-196ºC) and stored for several days. For thawing, straws were thawed at 30ºC for 10 s by gentle agitation in

On the other hand, post-thaw sperm quality tests were carred out to evaluate motility rate and duration of motility. For this aim, sperm motility rate and duration of motility values following cryopreservation in the same ionic extender containing 15% egg yolk were determined. Sperm was thawed at 25ºC, 35ºC or 45ºC for 5s, 15s or 25s and activated in 0.3%

Collected semen from the 5 males that showing >80 motility was pooled into equal aliquots according to the required semen volume and sperm density needed to eliminate effects of individual variability of the donors. Semen and extenders were kept at 4°C, then diluted at a ratio of 1:3 (semen/extender) with 3 different extenders containing 10% DMSO. Extender 1 contained 5.8 g/L NaCl, 0.2 g/L KCl, 0.22 g/L CaCl2, 0.04 g/L MgCl26H2O, 2.1 g/L NaHCO3, 0.04 g/L NaH2PO4.2H2O, 3.75 g/L glycine (Ravinder, et al. 1997). Extender 2 contained 300 mM glucose and 10% egg yolk pH:8 (Tekin et al. 2003) and extender 3 contained 4.68 g/L NaCl, 2.98 g/L KCl, 0.11 g/L CaCl2, 3.15 g/L Tris-HCl, pH:9 (Billard &

The diluted samples were drawn into 0.25 ml plastic straws (IMV, France) and were sealed with polyvinyl alcohol (PVA). Having been diluted, the samples were equilibrated for 10 min at 4ºC. After equilibration, the straws were placed on a styrofoam rack that floated on the surface of liquid nitrogen in a styrofoam box. The straws were frozen in liquid nitrogen vapour 3 cm above the surface of liquid nitrogen (-140ºC) for 10 min. After 10 min the straws were plunged into the liquid nitrogen (-196ºC) and stored for several days. For thawing, the straws were removed from liquid nitrogen and immersed in 30ºC water for 10 seconds. Thawed sperm was activated using 0.3% NaCl and observed under microscope for

For fertilization experiments, pooled eggs from 3 mature females were used to determine fertilization rates. Egg samples (about 100 eggs) were inseminated in dry Petri dishes with fresh sperm or frozen sperm immediately after thawing at a spermatozoa:egg ratio of 1×105: 1. Eggs were inseminated by the dry fertilization technique using a solution of 3 g urea and 4 g NaCl in 1 L distilled water. The sperm and eggs were slightly stirred for 30 min, washed with hatchery water (24ºC; 9 mg/l O2), and gently transferred to labeled Zuger glass incubators with running water (24°C) where they were kept until hatching (3-4 d). Living

was equilibrated for 10 min at 4°C.

NaCl and 1% NaHCO3.

Cosson, 1992).

water bath. Thawed sperm was activated using pond water.

**2.5 Experiment 2 - Ornamental koi carp (***Cyprinus carpio***)**

determination of spermatozoa motility and motility durations.

### **3.1 Fresh sperm quality parameters**

In brown trout fresh semen volumes were rather variable and ranged from 9 to 17 ml and mean volume was 12.6±4.28 mL. Motility values were ranged from 75% to 90%. Samples that motility values were below than 80% were not used for the cryopreservation experiments. The mean motility value of fresh sperm samples were 84.5±7.59%. Mean spermatozoa movement duration (s), sperm density x109/mL and pH values were achieved as 57.4±3.8 s, 24.8±4.62 x109/mL and 7.28±2.46 respectively.

In koi carp mean fresh semen volume, spermatoza motility, motility duration, spermatozoa density and pH values of the collected fresh milt samples were determined as 6.2±4.7 ml, 85.4±2.4%, 125.2±3.5 s, 22.8 x 109 mL-1 and 7.4±3.7, respectively.
