**3. Results and discussion**

#### **3.1 Effects of different cryoprotectants on fertility, hatchability, motility and survivability**

In the first trial with dilution ratio of 1:1 (sperm: extender) the effect of nature of cryoprotectants on the parameters measured was significant (p<0.05). Though dimethylsulphoxide+5% glucose+ PBS (DGP) was higher, DGP and DP gave the best results and was not significantly different (p>0.05) from each other but significantly different (p<0.05) from other cryoprotectants. It was followed by GP but not significantly different (p>0.05) from other cryoprotectants. Fertility also followed the same trend, but DGP was significantly different from DP from their LSD values followed by GP. GF gave the least result but significantly different (p<0.05) from other cryoprotectants. Related trend was also observed for other cryoprotectants but that of control was higher (p<0.05) than other treatments for each parameter. A similar trend was also observed for motility and survivability.

In Table 3, control (fresh semen), DGP and DP were compared. It is obvious that control has highest mean value (significantly different), this may be expected as the control was not passing through any treatments and processes.

In the second trial when the semen was diluted at ratio 1:20 (sperm: extender) a very close trend was observed which shows that the spermatozoa seems to be too much and probably wasted in the first trial. Besides control which was significantly different (p<0.05) for all the parameters, DGP gave the best result before DP, this may be explained by extracellular protection offered by the glucose, but the mean values are not significantly different (p>0.05) from each other. Fertility also related to hatchability followed the same trend with hatchability. In fertility, DP, DGP and GP were not significantly different from each other. However, the mean values are significantly different from each other for other parameters in

The data collected on the parameters, motility, fertility and hatchability was subjected to standard statistical analysis. The data collected were analyzed using analysis of variance (ANOVA) to find a level of significance at p < 0.05. The number of motile sperm counted per square of hemocytometer and percentage of motile sperm obtained after cryopreservation were subjected to Duncan's multiple range test to evaluate effects of types of cryopreservatives as well as egg clutch weights and sperm dilutions and period of cryopreservation on sperm motility. The data collected on motility, fertility and hatchability in the first trial were subjected to 2- way ANOVA at a significance level of p < 0.05. The bar charts and line chart showing the relationship between the period of refrigeration and the % motility for the various extenders were also employed for better understanding of the

The costs of chemicals and other consumables used for the study were listed in Table 5. The rates per gram or per ml were calculated to determine the effective cost per ml of the

In the first trial with dilution ratio of 1:1 (sperm: extender) the effect of nature of cryoprotectants on the parameters measured was significant (p<0.05). Though dimethylsulphoxide+5% glucose+ PBS (DGP) was higher, DGP and DP gave the best results and was not significantly different (p>0.05) from each other but significantly different (p<0.05) from other cryoprotectants. It was followed by GP but not significantly different (p>0.05) from other cryoprotectants. Fertility also followed the same trend, but DGP was significantly different from DP from their LSD values followed by GP. GF gave the least result but significantly different (p<0.05) from other cryoprotectants. Related trend was also observed for other cryoprotectants but that of control was higher (p<0.05) than other treatments for each parameter. A similar trend was also observed for motility and

In Table 3, control (fresh semen), DGP and DP were compared. It is obvious that control has highest mean value (significantly different), this may be expected as the control was not

In the second trial when the semen was diluted at ratio 1:20 (sperm: extender) a very close trend was observed which shows that the spermatozoa seems to be too much and probably wasted in the first trial. Besides control which was significantly different (p<0.05) for all the parameters, DGP gave the best result before DP, this may be explained by extracellular protection offered by the glucose, but the mean values are not significantly different (p>0.05) from each other. Fertility also related to hatchability followed the same trend with hatchability. In fertility, DP, DGP and GP were not significantly different from each other. However, the mean values are significantly different from each other for other parameters in

**3.1 Effects of different cryoprotectants on fertility, hatchability, motility and** 

**2.15 Statistical analysis** 

**2.16 Cost of production of cryopreserved semen** 

passing through any treatments and processes.

cryopreserved sperm (Table 6).

**3. Results and discussion** 

results.

**survivability** 

survivability.

decreasing order of C > DGP > DP > GP > GF. Generally, DGP gave the best followed by DP (without 5% glucose) while Glycerol in combination with Ginsburg fish ringer gave the lowest result.

A close means values were also discovered with trial 1 which indicate the results were better in trial 2, the further diluted semen which could supposedly be explained by addition of extenders.

The differences in fertility and hatchability with control and cryoprotectants tested may be due to the mild damage done to the spermatozoa during the process of lacerating the testes to extract the semen, and also due to the intracellular vitrification (Cryobiosystems, 2009)- a commonly occurring problem in the process of cryopreservation in liquid Nitrogen.
