**2.2 Result**

110 Current Frontiers in Cryobiology

where ∆*M*= the decrement in the glycerol molarity in the spermatozoa after each stepwise addition of the isotonic solution, *M*o = cryoprotective agent concentration (molarity) in the initial sperm suspension, n= total number of steps, *i*=*i*th-step addition, *V*o=original volume of cell suspension, \* *Vi*<sup>1</sup> = the total volume of cell suspension before the *i*th-step addition and *Vi*= volume of isotonic solution added into cell suspension at *it*h step. After n-1 step of addition , the cryoprotective agent concentration in the cell was diluted to ∆M. Then spermatozoa were transferred to isotonic conditions, which is the last (the nth) step removal

The final sperm suspensions (90 or 120 μl) were further diluted by adding 180 μl of TALP solution. The time interval between any two steps was ~0.5-1 min. The volume of diluent

Add 2000 μl of isotonic solution directly to 100 μl of cell suspension with 1.0 M glycerol Table 2. Procedures used in one-step and eight-step removal of 1.0 M glycerol from human

1. Add 2000 μl of sucrose buffer medium (TALP + sucrose, 600 mOsm to 100 μl of

Centrifuge the suspension (400 g for 7 min) and aspirate the supernatant.

were in contract with sucrose was 0.5 min before centrifugation.

Resuspend the cell pellet with 500 μl of isotonic TALP medium

Table 3. Procedures used in the two-step removal of 1.0 M glycerol from spermatozoa using

In the first step, glycerol was directly removed by transferring cells to a hyperosmotic medium (osmotic buffer, TALP with sucrose) containing no glycerol but only nonpermeating solutes (salts and sucrose), and in the second step spermatozoa in the osmotic

sperm suspension with 1.0 M glycerol. (The total length of time spermatozoa

where *i*=1, …, n-1 (17)

\* *VV V io k* <sup>1</sup> where *k*=1, …, i-1 (18)

(1) Stepwise add 14.3, 19, 26.6 and 40 μl of isotonic TALP medium to 100 μl of sperm suspension with 1.0 M glycerol; (2) centrifuge the supernatant; stepwise volume add 10, 20 and 60 μl of isotonic solution to the remaining 30 μl of sperm suspension. After the seven dilution steps, the glycerol concentration in the sperm suspension is 0.125 M. The final

suspension volume is 120 μl.

 \* 1 \*

1

Fixed-volume-step method Fixed-molarity-step method

1 <sup>1</sup> *i i o i V V nV V* 

of glycerol, see Table 2 fore examples.

Add 100 μl of isotonic TALP seven times to 100 μl of sperm suspension to achieve a final glycerol concentration of 0.125 M. After centrifugation, 710 μl of supernatant is taken off. The remaining

Eight-step dilution

cell suspension is 90 μl

One-step dilution

sucrose as an osmotic buffer

spermatozoa

*Approach 3*: Two-step dilution with an osmotic buffer

added in each step was calculated using equation [8] or [9]

The percentage of spermatozoa which maintained motility or plasma membrane integrity after each treatment was normalized to that of untreated, isotonic control samples and the data are so presented.

#### **Determination of osmotic injury as a function of sperm volume excursion**

Human spermatozoa were exposed for 5min to hyper- or hypo-osmotic solutions of sucrose and TALP salts ranging in concentration from 60 to 1200 mOsmol, and their motilities were then determined by CASA while still in those solutions. Figure 3 shows that sperm motilities dropped significantly when the osmolality was >50 mOsmol above or below isotonic (286 mOsmol). Motilities approached zero when the osmolalities were <200 or >600 mOsmol.

The next step was to compare these motilities with those observed after spermatozoa were transferred from these anisosmotic solutions back to near isotonic solutions. Figures 4 and 5 show the motilities as a function of time after transfer from hyperosmotic or from hypoosmotic exposures respectively. In both cases, the more the initial exposure departed from isotonicity, the greater the damage upon return to isotonicty. Most, or all, of the damage was evident in the first 30 s after the return, although in the case of transfer from hypertonic solutions to near isotonic, there was a further slight and gradual decline over the ensuing 30 min.

Figure 6 compares sperm motilities after a 5 min exposure to the various anisosmotic solutions before and after the return to near isotonic conditions. The reduction in the motilities of spermatozoa exposed to hypo-osmotic media was not affected by the return to isotonic media, but most of the apparent loss of motility of spermatozoa in hyperosmotic media of between 286 and 600 mOsmol was reversed when spermatozoa were returned to near isotonic. For example, although only 10% of spermatozoa were motile in 600 mOsmol

Prevention of Lethal Osmotic Injury to Cells

0

0

20

40

60

80

During Addition and Removal of Cryoprotective Agents: Theory and Technology 113

<sup>100</sup> *<sup>n</sup>*= 8

5 10 15 20

**TIME** (sec)

Fig. 5. Percent motility (mean±SEM, *n*=8) of human spermatozoa which were abruptly (onestep) returned to near isotonic conditions (273-284 mOsmol) after they had been exposed to different hyperosmotic conditions (TALP +water) for different periods of time. ■, 240 mOsmol; ○, 215 mOsmol;●, 190 mOsmol; ▽,143 mOsmol; ▼, 114 mOsmol; □, 90 mOsmol.

Fig. 6. A comparison of human sperm motility (% mean±SEM, *n*=8) after a 5 min exposure to the various hypo- and hyperosmotic solutions of non-permeating solutions before (○) and

after (□) the return to near isotonic conditions (273-343 mOsmol).

Fig. 3. Percent motility (mean±SEM, n=8) of human spermatozoa which were abruptly (onestep) exposed to different osmotic conditions for 5 min at 22°C.

Fig. 4. Percent motility (mean±SEM, n=8) of human spermatozoa which were abruptly (onestep) returned to near isotonic conditions (305-343 mOsmol) after they had been exposed to different hyperosmotic conditions (TALP +sucrose) for different periods of time. ▽, 600 mOsmol; ▼, 700 mOsmol;○, 900 mOsmol; ●, 1200 mOsmol.

Fig. 3. Percent motility (mean±SEM, n=8) of human spermatozoa which were abruptly (one-

Fig. 4. Percent motility (mean±SEM, n=8) of human spermatozoa which were abruptly (onestep) returned to near isotonic conditions (305-343 mOsmol) after they had been exposed to different hyperosmotic conditions (TALP +sucrose) for different periods of time. ▽, 600

step) exposed to different osmotic conditions for 5 min at 22°C.

mOsmol; ▼, 700 mOsmol;○, 900 mOsmol; ●, 1200 mOsmol.

Fig. 5. Percent motility (mean±SEM, *n*=8) of human spermatozoa which were abruptly (onestep) returned to near isotonic conditions (273-284 mOsmol) after they had been exposed to different hyperosmotic conditions (TALP +water) for different periods of time. ■, 240 mOsmol; ○, 215 mOsmol;●, 190 mOsmol; ▽,143 mOsmol; ▼, 114 mOsmol; □, 90 mOsmol.

Fig. 6. A comparison of human sperm motility (% mean±SEM, *n*=8) after a 5 min exposure to the various hypo- and hyperosmotic solutions of non-permeating solutions before (○) and after (□) the return to near isotonic conditions (273-343 mOsmol).

Prevention of Lethal Osmotic Injury to Cells

permeating solutes.

During Addition and Removal of Cryoprotective Agents: Theory and Technology 115

Since it has been shown that human spermatozoa behave as ideal osmometer over most of the range of osmolalities studied here (Du et al., 1993), a direct physical consequence of the exposures to anisosmotic conditions is major excursion in cell volume. The kinetics of volume excursion of spermatozoa in these hypo- and hyperosmotic solutions (containing only non-permeating solutes) were calculated and are plotted in Figure 8A and B

Fig. 8. (A) Calculated relative sperm volume (normalized to an isotonic sperm volume of 1) as a function of time after spermatozoa were one-step exposed to different hypo-osmotic solution containing non-permeating solutes. (B) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after the isotonic spermatozoa were one-step exposed to different hyperosmotic solutions containing non-

**Calculated volume excursions associated with exposures to anisosmotic solutions** 

Fig. 7. Membrane integrity (CFDA and propidium iodide stain) (% mean±SEM, *n*=8) of human spermatozoa which were abruptly (one-step) returned to near isotonic conditions (273-343 mOsmol) after they had been exposed to different anisosmotic conditions for 5 min.

solutions, 95% of spermatozoa were motile after return to isotonic media. The return to near isotonic became especially damaging, however, when the initial hyperosmotic concentration was >600 mOsmol.

Figure 7 shows that integrity of the plasma membrane of spermatozoa (as assessed by CFDA /propidium iodide) was substantially more resistant to wide excursions from isotonicity than was motility. Thus, >90% of those spermatozoa exposed to a 90 mOsmol salt solution retained intact plasma membrane after return to near isotonic, whereas <10% remained motile both before and after return to isotonic. Loss of plasma membrane integrity in 50% of the spermatozoa occurred only when spermatozoa were exposed to a 60 mOsmol solution, a figure that agrees with a previous report (Noiles et al, 1993); that loss occurs whether or not spermatozoa are returned to isotonic. This has been interpreted to represent lysis from the attainment of a cell volume in excess of that tolerated by the surface area of the plasma membrane.

Using light microscopy, morphological changes in sperm cells were observed during the exposure to anisosmotic solutions. In a portion of the spermatozoa, the tail region became configured as a 'zigzag' pattern when exposed to a hyper-osmotic solution. The pattern of sperm tail curling in hypo-osmotic solutions was osmolality dependent, which is consistent with a previous report (Jeyendran et al., 1984). In addition, the curling of sperm tails occurred not only when the isotonic spermatozoa were exposed to a relative hypo-osmotic condition. (For example, the shrunken spermatozoa in hyperosmotic solutions were returned to iso-osmotic conditions. Iso-osmolality was 'hypo' relative to a given hyper osmolality.) The tail curling was irreversible. The mechanism(s) behind the morphological change is not clearly understood.

Fig. 7. Membrane integrity (CFDA and propidium iodide stain) (% mean±SEM, *n*=8) of human spermatozoa which were abruptly (one-step) returned to near isotonic conditions (273-343 mOsmol) after they had been exposed to different anisosmotic conditions for 5 min.

was >600 mOsmol.

the plasma membrane.

change is not clearly understood.

solutions, 95% of spermatozoa were motile after return to isotonic media. The return to near isotonic became especially damaging, however, when the initial hyperosmotic concentration

Figure 7 shows that integrity of the plasma membrane of spermatozoa (as assessed by CFDA /propidium iodide) was substantially more resistant to wide excursions from isotonicity than was motility. Thus, >90% of those spermatozoa exposed to a 90 mOsmol salt solution retained intact plasma membrane after return to near isotonic, whereas <10% remained motile both before and after return to isotonic. Loss of plasma membrane integrity in 50% of the spermatozoa occurred only when spermatozoa were exposed to a 60 mOsmol solution, a figure that agrees with a previous report (Noiles et al, 1993); that loss occurs whether or not spermatozoa are returned to isotonic. This has been interpreted to represent lysis from the attainment of a cell volume in excess of that tolerated by the surface area of

Using light microscopy, morphological changes in sperm cells were observed during the exposure to anisosmotic solutions. In a portion of the spermatozoa, the tail region became configured as a 'zigzag' pattern when exposed to a hyper-osmotic solution. The pattern of sperm tail curling in hypo-osmotic solutions was osmolality dependent, which is consistent with a previous report (Jeyendran et al., 1984). In addition, the curling of sperm tails occurred not only when the isotonic spermatozoa were exposed to a relative hypo-osmotic condition. (For example, the shrunken spermatozoa in hyperosmotic solutions were returned to iso-osmotic conditions. Iso-osmolality was 'hypo' relative to a given hyper osmolality.) The tail curling was irreversible. The mechanism(s) behind the morphological

#### **Calculated volume excursions associated with exposures to anisosmotic solutions**

Since it has been shown that human spermatozoa behave as ideal osmometer over most of the range of osmolalities studied here (Du et al., 1993), a direct physical consequence of the exposures to anisosmotic conditions is major excursion in cell volume. The kinetics of volume excursion of spermatozoa in these hypo- and hyperosmotic solutions (containing only non-permeating solutes) were calculated and are plotted in Figure 8A and B

Fig. 8. (A) Calculated relative sperm volume (normalized to an isotonic sperm volume of 1) as a function of time after spermatozoa were one-step exposed to different hypo-osmotic solution containing non-permeating solutes. (B) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after the isotonic spermatozoa were one-step exposed to different hyperosmotic solutions containing nonpermeating solutes.

Prevention of Lethal Osmotic Injury to Cells

exposure to anisosmotic conditions for 1 min.

During Addition and Removal of Cryoprotective Agents: Theory and Technology 117

Fig. 10. Post-anisosmotic sperm motility recovery as a function of relative sperm volume (normalized to the isotonic sperm volume of 1) in different anisosmotic equilibrium states. Human spermatozoa were abruptly (one-step) returned to near isotonic conditions after

sperm membrane were simulated by computer. Figure 12 shows the calculated sperm volume excursion during a one—step or four-step addition of glycerol achieve a final 1.0 M glycerol concentration at 22 C using the FMS and FVS approaches respectively. From Figure 12, a one-step addition of glycerol to spermatozoa was predicted to cause ~20% sperm motility loss because the minimum volume which the cells would achieve during this glycerol addition was ~72% of the cells would achieve during this glycerol addition was ~72% of the original cell volume, i.e. below the LVL (75% or 0.75 ×isotonic sperm volume). In contrast, a four-step FMS glycerol addition was predicted to be able to prevent sperm loss (<5% loss). Figure 12 also shows a comparison between a four-step FVS and FMS approach. A four-step FVS method was predicted to cause a lower minimum volume than a four-step FMS method. From Figure 13, a one-step removal of 1.0 M glycerol was predicted to cause >70% motility loss, because the maximum cell volume during the glycerol removal was calculated to be in excess of 1.6 times the isotonic cell volume, which is much higher than the UVL (1.1×isotonic sperm volume). Figure 14 shows that a four- or six-step FMS removal procedure was predicted to reduce sperm motility loss significantly, but these still may cause >\*5 % motility loss, while an eight-step FMS removal was predicted to able to prevent sperm motility loss (<5% loss). Figure 13 also shows a comparison between an eight-step FMS and an eight-step FVS removal procedure. An eight-step FVS removal was predicted to cause a maximum cell swelling >1.2\* isotonic cell volume (>UVL), while the maximum cell volume during an eight-step FMS removal was predicted to be much lower than the UVL, indicating the eight-step FVS removal is not as good as an eight-step FMS. Based on the data presented in Figures 11-14, it was also found, from calculations, that human spermatozoa will

respectively, indicating that only a short time was required for human spermatozoa to achieve osmotic equilibration (<1 s for shrinking, and ≤30 s for swelling). Figure 8A and B also show the maximum or minimum volume of spermatozoa when they were osmotically equilibrated with each anisosmotic solution. Sperm equilibration volume as a function of extracellular osmolality is shown in Figure 9, which can be calculated using equation (6) (no cryoprotective agent) or obtained directly from Figure 8A and B. To obtain a high (>95%) motility recovery, the lowest and highest osmolalities which human spermatozoa can tolerate (Figures 4 and 5) were found to be close to 240 and 600 mOsmol respectively. At these two osmolalities, the corresponding cell volume at osmotic equilibrium were directly estimated (Figure 9) to be ~1.1 (for 240 mOsmol) and 0.75 (for 600 mOsmol) times the isotonic sperm volume, indicating that spermatozoa can only swell or shrink in a relatively narrow range to maintain high post-anisosmotic motility recovery. Based on Figure 4, 5 and 9, Figure 10 was plotted, which clearly shows the post-anisosmotic injury (motility loss) as a function of osmotic equilibrium volume of spermatozoa in anisosmotic solutions. Defining lower volume limit (LVL) and upper volume limit (UVL) as cell volumes at which 5% of motile spermatozoa may irreversibly lose their motility, or, reciprocally, 95% of spermatozoa maintain their motility, one can obtain the LVL and UVL values for human spermatozoa from Figure 10 as follows: LVL =0.75×isotonic sperm volume, UVL=1.10× isotonic sperm volume.

#### **Prediction of optimal protocols for glycerol addition/removal**

The kinetics of human sperm volume excursion during one-step addition and removal of 0.5-2.0 M glycerol were calculated using equations (6-9) and are shown in Figure 11A and B respectively. The higher the glycerol concentration, the longer the time period taken for sperm volume recovery and the greater the volume excursion.

Two different approaches, i.e. fixed-volume-step (FVS) and fixed-molarity-step (FMS), for the addition/removal of glycerol in spermatozoa were considered and used in the present example. Based equations (6-9), the kinetics of water and glycerol transport through the

Fig. 9. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) after spermatozoa were osmotically equilibrated to different anisosmotic conditions.

respectively, indicating that only a short time was required for human spermatozoa to achieve osmotic equilibration (<1 s for shrinking, and ≤30 s for swelling). Figure 8A and B also show the maximum or minimum volume of spermatozoa when they were osmotically equilibrated with each anisosmotic solution. Sperm equilibration volume as a function of extracellular osmolality is shown in Figure 9, which can be calculated using equation (6) (no cryoprotective agent) or obtained directly from Figure 8A and B. To obtain a high (>95%) motility recovery, the lowest and highest osmolalities which human spermatozoa can tolerate (Figures 4 and 5) were found to be close to 240 and 600 mOsmol respectively. At these two osmolalities, the corresponding cell volume at osmotic equilibrium were directly estimated (Figure 9) to be ~1.1 (for 240 mOsmol) and 0.75 (for 600 mOsmol) times the isotonic sperm volume, indicating that spermatozoa can only swell or shrink in a relatively narrow range to maintain high post-anisosmotic motility recovery. Based on Figure 4, 5 and 9, Figure 10 was plotted, which clearly shows the post-anisosmotic injury (motility loss) as a function of osmotic equilibrium volume of spermatozoa in anisosmotic solutions. Defining lower volume limit (LVL) and upper volume limit (UVL) as cell volumes at which 5% of motile spermatozoa may irreversibly lose their motility, or, reciprocally, 95% of spermatozoa maintain their motility, one can obtain the LVL and UVL values for human spermatozoa from Figure 10 as follows: LVL =0.75×isotonic sperm volume, UVL=1.10×

The kinetics of human sperm volume excursion during one-step addition and removal of 0.5-2.0 M glycerol were calculated using equations (6-9) and are shown in Figure 11A and B respectively. The higher the glycerol concentration, the longer the time period taken for

Two different approaches, i.e. fixed-volume-step (FVS) and fixed-molarity-step (FMS), for the addition/removal of glycerol in spermatozoa were considered and used in the present example. Based equations (6-9), the kinetics of water and glycerol transport through the

Fig. 9. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) after spermatozoa were osmotically equilibrated to different anisosmotic conditions.

isotonic sperm volume.

**Prediction of optimal protocols for glycerol addition/removal** 

sperm volume recovery and the greater the volume excursion.

Fig. 10. Post-anisosmotic sperm motility recovery as a function of relative sperm volume (normalized to the isotonic sperm volume of 1) in different anisosmotic equilibrium states. Human spermatozoa were abruptly (one-step) returned to near isotonic conditions after exposure to anisosmotic conditions for 1 min.

sperm membrane were simulated by computer. Figure 12 shows the calculated sperm volume excursion during a one—step or four-step addition of glycerol achieve a final 1.0 M glycerol concentration at 22 C using the FMS and FVS approaches respectively. From Figure 12, a one-step addition of glycerol to spermatozoa was predicted to cause ~20% sperm motility loss because the minimum volume which the cells would achieve during this glycerol addition was ~72% of the cells would achieve during this glycerol addition was ~72% of the original cell volume, i.e. below the LVL (75% or 0.75 ×isotonic sperm volume). In contrast, a four-step FMS glycerol addition was predicted to be able to prevent sperm loss (<5% loss). Figure 12 also shows a comparison between a four-step FVS and FMS approach. A four-step FVS method was predicted to cause a lower minimum volume than a four-step FMS method. From Figure 13, a one-step removal of 1.0 M glycerol was predicted to cause >70% motility loss, because the maximum cell volume during the glycerol removal was calculated to be in excess of 1.6 times the isotonic cell volume, which is much higher than the UVL (1.1×isotonic sperm volume). Figure 14 shows that a four- or six-step FMS removal procedure was predicted to reduce sperm motility loss significantly, but these still may cause >\*5 % motility loss, while an eight-step FMS removal was predicted to able to prevent sperm motility loss (<5% loss). Figure 13 also shows a comparison between an eight-step FMS and an eight-step FVS removal procedure. An eight-step FVS removal was predicted to cause a maximum cell swelling >1.2\* isotonic cell volume (>UVL), while the maximum cell volume during an eight-step FMS removal was predicted to be much lower than the UVL, indicating the eight-step FVS removal is not as good as an eight-step FMS. Based on the data presented in Figures 11-14, it was also found, from calculations, that human spermatozoa will

Prevention of Lethal Osmotic Injury to Cells

the diagrams.

sperm motility loss (<5%).

During Addition and Removal of Cryoprotective Agents: Theory and Technology 119

Fig. 13. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by one-step, eight fixed-molarity steps or eight fixed-volume steps. The estimates of percent motility recovery as a function of sperm relative volume were obtained from Figure 10 and are indicated in

rapidly achieve an osmotic equilibrium (within 15 s) during any stepwise addition or removal of glycerol. For example, from the calculations, human spermatozoa achieve osmotic equilibrium within 15 s after each step addition of glycerol by either one-step of four-step addition (Figure 12). This indicates that only a short time interval between steps of glycerol addition/removal is required for cells to achieve corresponding osmotic

In the analysis above, sperm osmotic injury (motility loss) caused by different glycerol addition/removal procedures has been predicted and a four-step FMS addition and an eight-step FMS removal of 1.0 M glycerol were found to be acceptable protocols to prevent

A two-step removal of cryoprotective agent from human spermatozoa using a nonpermeating solute as an osmotic buffer has been previously used to avoid osmotic injury in other cell types (Rowe et al., 1968; Leibo and Mazur, 1978; Watson, 1979). The steps involved in this approach are (i) the cryoprotective agent is directly removed and cell swelling is reduced by transferring cells with the cryoprotective agent to a hyperosmotic medium (osmotic buffer) of non-permeating solutes; and (ii) the cells in the osmotic buffer are rehydrated by directly transferring them to isotonic solution. Since current results showed that 600 mOsmol was the hyperosmotic upper tolerance limit for human spermatozoa to maintain 95% motility, the osmolality of the osmotic buffer medium should not exceed 600

equilibration volume after each step of glycerol addition and removal.

**Theoretical evaluation of two-step glycerol removal using an osmotic buffer** 

Fig. 11. (A) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after the isotonic sperm were exposed to different hyperosmotic glycerol solution isotonic with respect to non-permeating solutes (salt). (B) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after spermatozoa, which had been pre-equilibrated with different hyperosmotic glycerol solutions isotonic with respect to non-permeating solutes (salt), were one-step exposed to isotonic (286 mOsmol) saline solution without glycerol.

Fig. 12. (left) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1M glycerol was added to spermatozoa by either one-step or four fixed molarity steps. (right) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time 1M glycerol was added to spermatozoa by either one step or four fixed-volume steps. The estimates of percent motility recovery as a function of sperm relative volume were obtained from Figure 8 and are indicated in the diagrams.

Fig. 11. (A) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after the isotonic sperm were exposed to different hyperosmotic glycerol solution isotonic with respect to non-permeating solutes (salt). (B) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after spermatozoa, which had been pre-equilibrated with different hyperosmotic glycerol solutions isotonic with respect to non-permeating solutes (salt), were one-step exposed to

Fig. 12. (left) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1M glycerol was added to spermatozoa by either one-step or four fixed molarity steps. (right) Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time 1M glycerol was added to spermatozoa by either one step or four fixed-volume steps. The estimates of percent motility recovery as a function of sperm relative volume were obtained from Figure 8 and are indicated in the diagrams.

isotonic (286 mOsmol) saline solution without glycerol.

Fig. 13. Calculated relative sperm volume (normalized to the isotonic sperm volume of 1) as a function of time after 1 M glycerol was removed from spermatozoa by one-step, eight fixed-molarity steps or eight fixed-volume steps. The estimates of percent motility recovery as a function of sperm relative volume were obtained from Figure 10 and are indicated in the diagrams.

rapidly achieve an osmotic equilibrium (within 15 s) during any stepwise addition or removal of glycerol. For example, from the calculations, human spermatozoa achieve osmotic equilibrium within 15 s after each step addition of glycerol by either one-step of four-step addition (Figure 12). This indicates that only a short time interval between steps of glycerol addition/removal is required for cells to achieve corresponding osmotic equilibration volume after each step of glycerol addition and removal.

In the analysis above, sperm osmotic injury (motility loss) caused by different glycerol addition/removal procedures has been predicted and a four-step FMS addition and an eight-step FMS removal of 1.0 M glycerol were found to be acceptable protocols to prevent sperm motility loss (<5%).
