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

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


Means followed by different superscripts (lowercase for lines and uppercase for columns within the same sperm feature) are different (p<0.05). (mean±SE, n=3).

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

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%

Cryopreservation of Brown Trout

**Post-thaw motility (%)**

**Post-thaw motility longevity (s)** 

periods and activating agents.

**25ºC/5s**

**25ºC/15s**

**25ºC/25s**

**35ºC/5s**

**35ºC/15s**

**35ºC/25s**

**Thawing Temperatures (C) and Periods (s)**

**Extenders** 

of koi carp sperm.

**4. Discussion** 

(*Salmo trutta macrostigma*) and Ornamental Koi Carp (*Cyprinus carpio*) Sperm 299

Fig. 2. Post-thaw motility longevity (s) of brown trout sperm thawed at different degrees,

**45ºC/5s**

**45ºC/15s**

**45ºC/25s**

**0.3%NaCl 1%NaHCO3**

Effect of three different extenders containing 10% DMSO on the post-thaw motility and movement duration, fertilization and hatching rates of koi carp are shown in Table 3. Mean post-thaw motility of koi carp sperm was 75.3±6.4% while the best motility was determined as 85%. The overall mean fertilization rate was determined as 99.2±0.72 while the best fertilization rate was determined as 100%. The highest hatching rate was determined as 50% in all experimental groups. Motility features and hatching rates of cryopreserved koi carp

> **Post-thaw motlity duration (s)**

Table 3. Effect of different extenders on post-thaw motility, fertilization and hatching rates

Successful cryopreservation of fish spermatozoa depends on a range of factors including the collection of high quality sperm, equilibration conditions, choice of cryoprotectant medium, cooling/thawing regimes, and conditions for fertilization. Even though some general rules can be applied to any fish species, optimization of the protocol is needed for each individual species (Kopeika et al. 2007). Several factors have affected post-thaw quality of cryopreserved sperm from both brown trout (*Salmo trutta macrostigma*) and ornamental koi carp (*Cyprinus carpio*). The results obtained in the present study contribute significantly

**E1** 75.2±0.4a 27.5±1.2a 99.6±0.5 42.5±1.9b **E2** 78.6±0.7b 32.9±0.4b 99.7±0.5 46.2±0.7c **E3** 72.3±0.2a 25.2±0.6a 98.3±1.2 37.4±0.2a **Control** - - 99.8±0.2 86.2±0.4d

**Fertilization rates (%)** 

**Hatching rates (%)** 

sperm was statistically different between the experimental groups (p<0.05).

Means followed by different superscripts are different (p<0.05). (mean±SE, n=3).

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

**Post-thaw motility (%)** 


egg yolk as 54.2±3.46 s. Differences between the means of motility durations were significant (P<0.05). (Table 2).

Means followed by different superscripts (lowercase for lines and uppercase for columns within the same sperm feature) are different (p<0.05). (mean±SE, n=3).

Table 2. Post-thaw longevity (s) of brown trout sperm cryopreserved with different cryoprotectants.

Sperm motility rate (Figure 1) and longevity of motility (Figure 2) values following cryopreservation in the 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.

Fig. 1. Post-thaw motility (%) of brown trout sperm thawed at different degrees, periods and activating agents.

Post-thaw sperm motility rates were affected by thawing rates and activation agents and ranged from 25% to 50%. Also, the activating agents affected the duration of motility. All sperm samples triggered in 1% NaHCO3 were motile for a longer period (32-57 s) compared with samples triggered in 0.3% NaCl (24-53 s). Differences between the post-thaw motility and longevity values were significant (P<0.05).

Fig. 2. Post-thaw motility longevity (s) of brown trout sperm thawed at different degrees, periods and activating agents.

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

Effect of three different extenders containing 10% DMSO on the post-thaw motility and movement duration, fertilization and hatching rates of koi carp are shown in Table 3. Mean post-thaw motility of koi carp sperm was 75.3±6.4% while the best motility was determined as 85%. The overall mean fertilization rate was determined as 99.2±0.72 while the best fertilization rate was determined as 100%. The highest hatching rate was determined as 50% in all experimental groups. Motility features and hatching rates of cryopreserved koi carp sperm was statistically different between the experimental groups (p<0.05).


Means followed by different superscripts are different (p<0.05). (mean±SE, n=3).

Table 3. Effect of different extenders on post-thaw motility, fertilization and hatching rates of koi carp sperm.
