**2.1 Broodstock management**

The experiments were carried out spawning season of the brown trout (*Salmo trutta macrostigma*) and koi carp (*Cyprinus carpio*). In the pre-spawning period the mature bown trouts were kept seperately in small ponds under constant environmental conditions. The water temperature ranged 8-10°C during the spawning period. During the experiment, fish were kept under natural photoperiod. Mean water temperature and dissolved oxygen of the broodstock ponds were 8.7±2.46°C and 9.2±7.2 ppm respectively.

The koi carp broodstock was collected from wintering ponds by seining and transported into the hatchery 48 h prior to gamete collection. In the hatchery, male and female broodfish were held separetely in shadowed tanks (V=1000 L) supplied with continuously (2.5 L min-1) well-aerated water of 24ºC. Brown trout and koi carp broodstock were not fed during the experiments.

#### **2.2 Gamete collection**

Sperm was collected by gently hand-stripping without anesthesia from mature 10 brown trout males. For koi carp cryopreservation experiments, semen was collected from 5

Bozkurt, 2008). Thawing temperature and duration are also critical factors in the survival of cryopreserved sperm cells (Morris, 1981). Optimal freezig/thawing procedures have not been reported for *Salmo trutta macrostigma* sperm. So, in the present study three different

For this reason, there is a need to improve techniques on gamete storage and evaluation of sperm quality to facilitate optimization of controlled reproduction in fish (Alavi & Cosson, 2005). Important parameters for cryopreservation include type of extenders and cryoprotectants, dilution ratios, freezing/thawing rates and fertilization rates (Bozkurt et al.

*Salmo trutta macrostigma* is a salmonid species occurring in inland water habitats of Southern Europe, Western Asia, Northern Africa and Anatolia (Geldiay & Balik, 1988). It is also critically endangered fish species in inland waters because of illegal shing, overshing, and other environmental changes, including hydroelectric plants and pollution. For this reason a biological conservation program has been considered for *Salmo trutta macrostigma* in Turkey. On the other hand, ornamental koi carp is evaluated by its colour and have been used in the selecive propagation programs. These brightly colored koi carps are the result of selective breeding of wild carp. Over centuries a range of pleasing colors, patterns and shapes have been developed for this valuable species. Therefore, reliable methods for brown trout and koi carp sperm cryopreservation could benefit both aquaculture application and

Therefore, the present study was conducted in order to examine the effect of ionic extenders combined with different cryoprotectants at different ratios and to test the effect of different thawing temperatures and thawing periods on the post-thaw sperm quality of brown trout (*Salmo trutta macrostigma*) and koi carp (*Cyprinus carpio*) and development of a

The experiments were carried out spawning season of the brown trout (*Salmo trutta macrostigma*) and koi carp (*Cyprinus carpio*). In the pre-spawning period the mature bown trouts were kept seperately in small ponds under constant environmental conditions. The water temperature ranged 8-10°C during the spawning period. During the experiment, fish were kept under natural photoperiod. Mean water temperature and dissolved oxygen of the

The koi carp broodstock was collected from wintering ponds by seining and transported into the hatchery 48 h prior to gamete collection. In the hatchery, male and female broodfish were held separetely in shadowed tanks (V=1000 L) supplied with continuously (2.5 L min-1) well-aerated water of 24ºC. Brown trout and koi carp broodstock were not fed during the

Sperm was collected by gently hand-stripping without anesthesia from mature 10 brown trout males. For koi carp cryopreservation experiments, semen was collected from 5

cryopreservation protocol for sperm of this commercially valuable two species.

broodstock ponds were 8.7±2.46°C and 9.2±7.2 ppm respectively.

thawing temperatures and thawing durations were also tested related to motility.

2005).

conservation of biodiversity.

**2. Materials and methods 2.1 Broodstock management** 

experiments.

**2.2 Gamete collection** 

anesthetized (0.1 g/l MS 222) males by manual abdominal stripping 12 h after a single injection of 2 mg/kg of carp pituitary extract (CPE) at 20-22 ºC water temperature. Eggs were collected by hand stripping 10-12 h after a double injection of 3.5 mg/kg of CPE. The first injection, 10% (0.35 mg/kg) CPE was given 10 h before the second (3.15 mg/kg).

For sperm collection, the urogenital papilla's of mature male fishes were carefully dried and sperm was hand-stripped directly into test tubes. Following sperm collection, the tubes containing sperm were placed in a styrofoambox containing crushed ice (4ºC). Contamination of sperm with water, urine or faeces was carefully avoided. Sperm was transported to the laboratory within 15 min. For collection of eggs from koi carps, females were wiped dry, stripped by gentle abdominal massage and the eggs from each female were collected in a dry metal bowl. Eggs were checked visually and only those lots of homogenous shape, colour and size were used in the fertilization experiments.

#### **2.3 Determination of fresh sperm quality parameters**

Motility was estimated subjectively using light microscope (Olympus, Japan) with a x400 magnification. Samples were activated by mixing 1 μl of sperm with 20 μl activation solution (0.3% NaCl) on a glass slide. The percentage of motility was defined as the percentage of spermatozoa moving in a forward motion every 20% motile increment (i.e., 0, 20%, 40%, 60%, 80%, and 100%) (Vuthiphandchai & Zohar, 1999). Motility measurements were performed within 15 s. after activation. Sperm cells that vibrated in place were not considered to be motile. Sperm motility was estimated with three replicates of samples. For cryopreservation experiments, samples below 80% motile spermatozoa were discarded. Duration of sperm motility was determined using a sensitive chronometer (sensitivity: 1/100 s) by recording the time following addition of the activation solution to the sperm samples.

Spermatozoa density was determined according to the haemacytometric method. Sperm was diluted at ratio of 1:1000 with Hayem solution (5g Na2SO4, 1g NaCl, 0.5g HgCl2, 200 mL bicine) and density was determined using a 100 μm deep Thoma haemocytometer (TH-100, Hecht-Assistent, Sondheim, Germany) at 400x magnification with Olympus BX50 phase contrast microscope (Olympus, Japan) and expressed as spermatozoa x109 mL-1 (three replicates). Counting chambers were always kept in a moist atmosphere for at least 10 min before cell counting. Sperm pH was measured using indicator papers (Merck, 5.5-9) within 30 min of sampling.

#### **2.4 Experiment 1 - Brown trout (***Salmo trutta macrostigma***)**

Collected sperm from 10 males that showing >80 motility was pooled into equal aliquots according to the required semen volume and sperm density to eliminate effects of individual variability of gamete donors. Semen and extenders were kept at 4°C prior to dilution. Pooled semen was diluted at 1:3 ratio (semen/extender) with extender containing 4.68 g l- NaCl, 2.98 g l- KCl, 0.11 g l- CaCl2 and Trizma-HCl 3.15 g l- in distilled water; pH 9.0 (Billard & Cosson, 1992). The extender contained methanol and egg yolk at ratios of 5%, 10% and 15% separately. Dilution of semen with extender resulted in sperm concentrations of around 2.5x109 cells/ml extender that was enough to avoid damage due to sperm

Cryopreservation of Brown Trout

**2.6 Statistical analysis** 

**3.1 Fresh sperm quality parameters** 

**3. Results** 

Methanol (5%)

cryoprotectants.

Methanol (10%)

same sperm feature) are different (p<0.05). (mean±SE, n=3).

(*Salmo trutta macrostigma*) and Ornamental Koi Carp (*Cyprinus carpio*) Sperm 297

and dead eggs were counted in each incubator during incubation and dead eggs were removed. When the fertilized eggs developed to embryos at the gastrula stage, the fertilization rate (number of gastrula stage embryos/number of total eggs) was calculated.

Results are presented as means±SE. Differences between parameters were analyzed by repeated analysis of variance (ANOVA). Significant means were subjected to a multiple comparison test (Duncan) for post-hoc comparisons at a level of α=0.05. All analyses were

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

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,

Post-thaw motility of sperm cryopreserved in ionic extender containing two different cryoprotectants at three different ratios is shown in Table 1. There were significant effect of cryoprotectants on motility rates. Sperm samples cryopreserved in the extenders containing egg yolk yielded greater post-thaw motility rates than methanol containing extenders. Sperm frozen with extender containing 15% egg yolk had the highest post-thaw motility.

10.6±4.57Af 15.2±5.80Ae 17.4±4.72Ae 40.5±3.27Aab 42.3±6.1Aa 45.3±4.27Aa 7.5±2.69Ae 9.6±3.37Be 12.3±5.24Ad 30.6±2.86Bb 35.4±4.17Abab 40.2±5.36Aba 5.4±2.73Ae 7.2±3.79Be 10.5±4.27Abd 25.7±4.69BCbc 30.2±5.29Bb 37.8±8.29Ba

Means followed by different superscripts (lowercase for lines and uppercase for columns within the

It was observed that a decrease in motility duration occurred following cryopreservation. The longest post-thaw motility longevity was also achieved with extender containing 15%

Table 1. Post-thaw motility (%) of brown trout sperm cryopreserved with different

Egg Yolk (5%)

Egg Yolk (10%)

Egg Yolk (15%)

carried out using SPSS 10 for Windows statistical software package.

as 57.4±3.8 s, 24.8±4.62 x109/mL and 7.28±2.46 respectively.

85.4±2.4%, 125.2±3.5 s, 22.8 x 109 mL-1 and 7.4±3.7, respectively.

**3.2. Experiment 1 - Brown trout (***Salmo trutta macrostigma***)**

Differences between the post-thaw motility values were significant (P<0.05).

Methanol (15%)

compression during freezing and thawing (Lahnsteiner, 2000). Following sperm suspension was equilibrated for 10 min at 4°C.

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 water bath. Thawed sperm was activated using pond water.

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% NaCl and 1% NaHCO3.

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

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 & Cosson, 1992).

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 determination of spermatozoa motility and motility durations.

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 and dead eggs were counted in each incubator during incubation and dead eggs were removed. When the fertilized eggs developed to embryos at the gastrula stage, the fertilization rate (number of gastrula stage embryos/number of total eggs) was calculated.
