**3. Results**

#### **3.1 Effects of direct and indirect exposure of sperm to liquid nitrogen on the post thawing sperm motility**

In the first study, we compared sperm motility after freezing with the Method I and Method II, i.e. direct and indirect immersion of sperm freezing devices (**Figure 1**) to liquid nitrogen and found that no sperm were motile if the devices were directly immersed to liquid nitrogen, while indirect immersion had high post thawing sperm motility. As shown in **Table 1**, the original motility was 85.0 ± 1.0%, and post thawing motilities were 79.7 ± 1.5% and 77.7 ± 2.1% with SpermVD and Microdevice, respectively. The recovery rates (post thawing motility/original motility) of motility were 93.7 ± 0.7% and 91.3 ±1.4% with SpermVD and Microdevice, respectively. No statistical differences were found between the devices (**Table 1**). Based on these findings, all following experiments were done in the SpermVD with the Method II.

After temperature changes were examined during direct and indirect immersion of devices to liquid nitrogen, as shown in **Figure 2**, we found that direct immersion has a 107°C/second cooling rate, or 2 seconds from 25°C to −190°C, while indirect


*\* Method I: Direct immersion of sperm freezing devices to liquid nitrogen.*

*\*\*Method II: Sperm freezing devices were placed cryogenic storage vial, vials were sealed and then immersed to liquid nitrogen.*

**\*\*\****Percentage of post thawing sperm motility/original motility.*

*abValues are significantly different in the same column, P < 0.00001.*

#### **Table 1.**

*Effects of direct and indirect exposure of sperm to liquid nitrogen on the post thawing sperm motility.*

*A Method for Small Number of Human Sperm Cryopreservation DOI: http://dx.doi.org/10.5772/intechopen.98674*

#### **Figure 2.**

*Temperature changes during direct and indirect immersion of sperm freezing devices to liquid nitrogen. Times were recorded at 0, 2, 5, 10, 30, 60, 90 and 120 seconds during cooling from room temperature (25°C) to* −*190°C.*

immersion has slow cooling rates with a 2 min of total cooling time from 25°C to −190°C. However, the cooling rates were further examined during this period and we found that it was 1.2°C/second during the first 2 seconds (25°C to 22.6°C), 4.6°C/ second from 22.6°C to 8.7°C, 7.1°C/second from 8.7°C to −27°C, 4.03°C/second from −27°C to −107.6°C, 1.87°C/second from −107.6°C to −163.8°C, 0.66°C/second from −163.8°C to −183.6°C and 0.22°C/second from −183.6°C to −190°C. The temperature and time were not examined after the temperature reached to −190°C as it showed very slow changes after −190°C.

#### **3.2 Effects of freezing volumes on the post warming sperm motility**

As shown in **Table 2**, we found that there were no significant differences in the post thawing sperm motility between 1, 2 and 5 μl of freezing volumes. The original motility was 95.6 ± 1.7%, and post thawing motilities were 83.5 ± 5.5%, 88.9 ± 1.3%, and 86.4 ± 5.0%, respectively. The recovery rates of motility were 87.4 ± 6.2, 93.0 ± 1.9 and 90.5 ± 5.2%, respectively.


*Method II and SpermVD were used to freeze sperm.*

**\*\****Percentage of post thawing sperm motility/original motility.*

#### **Table 2.**

*Effects of freezing volumes on the post warming sperm motility\* .*

#### **3.3 Effects of equilibration times on post thawing sperm motility**

As shown in **Table 3**, we found that equilibration of 1, 2 and 5 minutes of sperm in the freezing solution had similar post thawing sperm motility. Original motility was 94 ± 2%, and post thawing motilities were 88.3 ± 2.1, 85.4 ± 1.6 and 84.1 ± 0.9%, respectively. The recovery rates of motility were 94.0 ± 1.6, 90.9 ± 3.0 and 89.5 ± 2.4%, respectively.


*\*\*Sperm equilibration time in the freezing solution before freezing.*

**\*\*\****Percentage of post thawing sperm motility/original motility.*

**Table 3.**

*Effects of equilibration times on post thawing sperm motility\* .*

## **3.4 Effects of freezing solution ratios on the post warming sperm motility**

In this experiment, no differences were observed in the post thawing sperm motility between different ratios of sperm freezing medium (1:1, 2:1 and 5:1). As shown in **Table 4**, the original sperm motility was 91.4 ± 2.4%, and post thawing motility was 83.0 ± 3.7, 86.8 ± 2.8 and 85.2 ± 1.8%, respectively. The recovery rates of sperm motility were 90.9 ± 5.4, 94.6 ± 3.7 and 93.1 ± 4.2%, respectively. However, if sperm were frozen in the solution without sperm freezing medium (0:1), only 6.2 ± 1.1% motility, or 6.8 ± 1.2% of recovery rate of motility was obtained (**Table 4**).


*\* Method II and SpermVD were used to freeze sperm.*

*\*\*Ratio of sperm freezing solution to sperm washing medium.*

**\*\*\****Percentage of post thawing sperm motility/original motility.*

*abValues are significantly different in the same column, P < 0.00001.*

#### **Table 4.**

*Effects of freezing solution ratios on the post warming sperm motility\* .*
