**Technological procedure, shortly**

*Vitrification.* Prior to vitrification, spermatozoa were processed by swim-up technique with subsequent dilution with cryoprotectant medium according to Isachenko (Isachenko et al.,

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 55

(Arrows) line, (a) aspiration of spermatozoa suspension in straw, (b, e) flame-sealing of straw,

Fig. 6. Schema of human spermatozoa vitrification using 0.5 mL straws.

from straw.

(c) cooling of straw, (d) warming of straw, (f) cutting of straw, (g) expelling of spermatozoa suspension

33258 staining technique (Kay et al. 1994). The results of that comparative investigation have shown that motility of spermatozoa vitrified in large volume (500 µL) in absence of permeable cryo-protectants displayed statistically higher levels of motility as compared to slow conventional freezing (76.0 +4.7 % *vs* 52.0 + 3.9 %, respectively, P<0.05; in fresh 85.0 +

2008). Diluted suspensions were maintained at room temperature for 5 minutes before the cooling procedure. The packaging of spermatozoa for aseptic vitrification was performed in the following way. Spermatozoa suspensions were cooled in 0.5 mL plastic CBS straws (CryoBio System, Paris, France) (Figure 6). The straw was labeled with asterisk (1 cm from the inner end of cotton-polyvinil plunge, arrows on Figures 6a-g). The straw was filled up to asterisk with 0.5 mL of spermatozoa suspension by aspiration (Figure 6a). Then the filled straw was expelled from the tube while aspiration of air continued. Subsequently, when the suspension reached the polyvinyl plunge, the polymerization of polyvinyl was initiated due to humidification. After aspiration was completed, and the top end of straw was sealed by polymerized polyvinyl, straw was hermetically heat-sealed at both sides using flame of alcohol burner and forceps (Figures 6b,e). The hermetically sealed straw with spermatozoa was allowed to cool briefly (~ 2 seconds). This procedure ensured that spermatozoa at any time were not in contact with the heat-sealing area. Alternatively, any commercial equipment (with exception of ultrasound equipment) could be used for thermo-hermetic sealing. The straws were immersed into liquid nitrogen in horizontal position (approximately for 8 seconds) (Figure 6c) and stored there at least for 24 hours before use.

*Warming.* The warming up of spermatozoa is achieved by immersing straw with vitrified spermatozoa into water bath at 42°C and dangling it gently in water for 20 seconds (Figure 6 d). After warming, the residual fluid was removed from the straw with paper towel, and straw disinfected with 70% ethanol. The heat-sealed part of straw (opposite to the cottonpolyvinyl plunge) was cut off with sterile scissors, and the aspirator was connected with the straw (Figure 6f). A low differential negative pressure was applied by aspiration. That ensured that after subsequent cutting of the cotton-polyvinyl plunge fluid was not leaking out (Figure 6f). Finally, the suspension was expelled from the straw (Figure 6g) for immediate evaluation of sperm quality, loading into catheter and intrauterine insemination.

The results were compared to slow frozen spermatozoa. For this purpose the Freezing Medium TYB, IrvineScientific, with 12 % (v/v) glycerol and 20 % (v/v) egg yolk were used. The suspension of swim up-prepared spermatozoa was 1:2 diluted with freezing medium (to achieve the concentration of 0.5 x 106 spermatozoa / mL and equilibrated at room temperature for 10 minutes then the 500 µL of spermatozoa suspension was packaged into 0.5 mL plastic straws (Cryo Bio System, Paris, France), the straws were sealed from both sides, kept in horizontal position at 4 °C for 30 minutes and put in the horizontal position into liquid nitrogen vapor (-80 °C, 10 cm over liquid nitrogen surface), kept for 30 minutes and finally placed into liquid nitrogen where they were stored minimum 24 hours until evaluation. For thawing of samples, the straws were taken from liquid nitrogen, hold in air for 30 seconds, immersed into 37°C water bath in horizontal position and hold in this bath for 20 seconds until ice melted. After thawing, 10 mL of basic (HTF-HSA) medium was added to thawed sample and centrifuged for 5 minutes at 340g. The supernatant was removed and pellet resuspended with the same basic medium in order to obtain a final concentration of 0.5 x 106 spermatozoa/mL. The changes in following physiological and morphological parameters of thirty minutes after warming of fresh, vitrified and conventional frozen spermatozoa were investigated for progressive motility (WHO, 1999); cytoplasmic membrane integrity (CMI) with applying of a LIVE/DEAD sperm viability kit, which is used to stain nucleic acid probe molecular (SYBR-14 dye) and propidium iodide (IP) and Acrosomal membrane integrity (AMI). The acrosome-reacted, and capacitated spermatozoa were detected using the double fluorescence chlortetracycline (CTC)-Hoechst

2008). Diluted suspensions were maintained at room temperature for 5 minutes before the cooling procedure. The packaging of spermatozoa for aseptic vitrification was performed in the following way. Spermatozoa suspensions were cooled in 0.5 mL plastic CBS straws (CryoBio System, Paris, France) (Figure 6). The straw was labeled with asterisk (1 cm from the inner end of cotton-polyvinil plunge, arrows on Figures 6a-g). The straw was filled up to asterisk with 0.5 mL of spermatozoa suspension by aspiration (Figure 6a). Then the filled straw was expelled from the tube while aspiration of air continued. Subsequently, when the suspension reached the polyvinyl plunge, the polymerization of polyvinyl was initiated due to humidification. After aspiration was completed, and the top end of straw was sealed by polymerized polyvinyl, straw was hermetically heat-sealed at both sides using flame of alcohol burner and forceps (Figures 6b,e). The hermetically sealed straw with spermatozoa was allowed to cool briefly (~ 2 seconds). This procedure ensured that spermatozoa at any time were not in contact with the heat-sealing area. Alternatively, any commercial equipment (with exception of ultrasound equipment) could be used for thermo-hermetic sealing. The straws were immersed into liquid nitrogen in horizontal position (approximately for 8 seconds) (Figure 6c) and stored there at least for 24 hours before use. *Warming.* The warming up of spermatozoa is achieved by immersing straw with vitrified spermatozoa into water bath at 42°C and dangling it gently in water for 20 seconds (Figure 6 d). After warming, the residual fluid was removed from the straw with paper towel, and straw disinfected with 70% ethanol. The heat-sealed part of straw (opposite to the cottonpolyvinyl plunge) was cut off with sterile scissors, and the aspirator was connected with the straw (Figure 6f). A low differential negative pressure was applied by aspiration. That ensured that after subsequent cutting of the cotton-polyvinyl plunge fluid was not leaking out (Figure 6f). Finally, the suspension was expelled from the straw (Figure 6g) for immediate evaluation of sperm quality, loading into catheter and intrauterine insemination. The results were compared to slow frozen spermatozoa. For this purpose the Freezing Medium TYB, IrvineScientific, with 12 % (v/v) glycerol and 20 % (v/v) egg yolk were used. The suspension of swim up-prepared spermatozoa was 1:2 diluted with freezing medium (to achieve the concentration of 0.5 x 106 spermatozoa / mL and equilibrated at room temperature for 10 minutes then the 500 µL of spermatozoa suspension was packaged into 0.5 mL plastic straws (Cryo Bio System, Paris, France), the straws were sealed from both sides, kept in horizontal position at 4 °C for 30 minutes and put in the horizontal position into liquid nitrogen vapor (-80 °C, 10 cm over liquid nitrogen surface), kept for 30 minutes and finally placed into liquid nitrogen where they were stored minimum 24 hours until evaluation. For thawing of samples, the straws were taken from liquid nitrogen, hold in air for 30 seconds, immersed into 37°C water bath in horizontal position and hold in this bath for 20 seconds until ice melted. After thawing, 10 mL of basic (HTF-HSA) medium was added to thawed sample and centrifuged for 5 minutes at 340g. The supernatant was removed and pellet resuspended with the same basic medium in order to obtain a final concentration of 0.5 x 106 spermatozoa/mL. The changes in following physiological and morphological parameters of thirty minutes after warming of fresh, vitrified and conventional frozen spermatozoa were investigated for progressive motility (WHO, 1999); cytoplasmic membrane integrity (CMI) with applying of a LIVE/DEAD sperm viability kit, which is used to stain nucleic acid probe molecular (SYBR-14 dye) and propidium iodide (IP) and Acrosomal membrane integrity (AMI). The acrosome-reacted, and capacitated spermatozoa were detected using the double fluorescence chlortetracycline (CTC)-Hoechst

(Arrows) line, (a) aspiration of spermatozoa suspension in straw, (b, e) flame-sealing of straw, (c) cooling of straw, (d) warming of straw, (f) cutting of straw, (g) expelling of spermatozoa suspension from straw.

Fig. 6. Schema of human spermatozoa vitrification using 0.5 mL straws.

33258 staining technique (Kay et al. 1994). The results of that comparative investigation have shown that motility of spermatozoa vitrified in large volume (500 µL) in absence of permeable cryo-protectants displayed statistically higher levels of motility as compared to slow conventional freezing (76.0 +4.7 % *vs* 52.0 + 3.9 %, respectively, P<0.05; in fresh 85.0 +

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 57

All rates in respective groups are significantly different (P<0.05) instead columns marked with asterisks

Fig. 8. Cytoplasmic and acrosomal membranes integrity as well as cryo-induction of capacitation of human spermatozoa after conventional freezing and vitrification.

(P>0.1).

5.1%) as well as after 24 and 48 hours in vitro culture (Figure 7). It was observed, that higher rates of membrane integrity (Figure 8) were achieved in vitrified sperm as compare to slow conventional freezing (54.0 ± 5.0 % vs 28.3 ± 3.5 %, respectively, P<0.05), but lower then in non-treated fresh control (98.2 ± 0.5 %, P<0.05). The effect of two procedures used for cryopreservation on sperm functional state as assessed by CTC staining is shown on Figure 8. There was a statistically significant difference between percentages of spermatozoa with intact acrosome after vitrification as compared to conventional freezing (44.4 ± 4.5 % *vs* 30.0 ± 3.9 %, respectively, P<0.05), but statistically lower then in fresh non-treated samples (95.4 ± 5.0 %; P<0.05). There were no statistically significant difference between percentages of sperm identified as 'capacitated' in CTC staining after vitrification as compared to conventional freezing (10.0 ± 1.8 % vs 11.0 ± 1.1 %, respectively, P <0.01), but significantly higher in fresh non-treated control (4.0 ± 0.2%, P<0.05). Described technology has a massive potential for applications in reproductive assisted procedures (ICSI, IVF and IUI) not only because of its simplicity but also because this procedure can effectively protect these cells from cryo-injures, at a level at least comparable to conventional freezing as judged by basic parameters of spermatozoa quality.

This cryoprotectant-free vitrification technology for the cryopreservation of spermatozoa instead traditional slow freezing with permeable cryoprotectants is already used in following centers: our university maternal hospital (www.uniklinik-ulm.de): IVF Centers in Temuco, Chile (about 200 IUI cycles/year) and in Ulm, Germany (www.kinderwunschulm.de) (about 1,000 IVF cycles/year). First successful pregnancies and birth of healthy babies after insemination of vitrified spermatozoa has been recently achieved with vitrified spermatozoa (Isachenko et al., 2011d; Sanchez et al., 2011a).

In conclusion, a basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.

All rates in respective groups are significantly different (P<0.05) instead columns marked with asterisks (P>0.1).

5.1%) as well as after 24 and 48 hours in vitro culture (Figure 7). It was observed, that higher rates of membrane integrity (Figure 8) were achieved in vitrified sperm as compare to slow conventional freezing (54.0 ± 5.0 % vs 28.3 ± 3.5 %, respectively, P<0.05), but lower then in non-treated fresh control (98.2 ± 0.5 %, P<0.05). The effect of two procedures used for cryopreservation on sperm functional state as assessed by CTC staining is shown on Figure 8. There was a statistically significant difference between percentages of spermatozoa with intact acrosome after vitrification as compared to conventional freezing (44.4 ± 4.5 % *vs* 30.0 ± 3.9 %, respectively, P<0.05), but statistically lower then in fresh non-treated samples (95.4 ± 5.0 %; P<0.05). There were no statistically significant difference between percentages of sperm identified as 'capacitated' in CTC staining after vitrification as compared to conventional freezing (10.0 ± 1.8 % vs 11.0 ± 1.1 %, respectively, P <0.01), but significantly higher in fresh non-treated control (4.0 ± 0.2%, P<0.05). Described technology has a massive potential for applications in reproductive assisted procedures (ICSI, IVF and IUI) not only because of its simplicity but also because this procedure can effectively protect these cells from cryo-injures, at a level at least comparable to conventional freezing as judged by basic

This cryoprotectant-free vitrification technology for the cryopreservation of spermatozoa instead traditional slow freezing with permeable cryoprotectants is already used in following centers: our university maternal hospital (www.uniklinik-ulm.de): IVF Centers in Temuco, Chile (about 200 IUI cycles/year) and in Ulm, Germany (www.kinderwunschulm.de) (about 1,000 IVF cycles/year). First successful pregnancies and birth of healthy babies after insemination of vitrified spermatozoa has been recently achieved with vitrified

In conclusion, a basic protection from cryo-injury can be achieved for human spermatozoa using the novel technology of aseptic cryoprotectant-free vitrification in large volumes.

All rates in respective groups are significantly different (P<0.05) instead columns marked with asterisks

Fig. 7. Motility of human spermatozoa after conventional freezing and vitrification.

parameters of spermatozoa quality.

(P>0.1).

spermatozoa (Isachenko et al., 2011d; Sanchez et al., 2011a).

All rates in respective groups are significantly different (P<0.05) instead columns marked with asterisks (P>0.1).

Fig. 8. Cytoplasmic and acrosomal membranes integrity as well as cryo-induction of capacitation of human spermatozoa after conventional freezing and vitrification.

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 59

Took into account all mentioned above in our investigation (Sánchez et al., 2011b) to decrease the sensitivity of dog spermatozoa to different manipulations before cryopreservation we have chosen the Human tubal fluid (HTF, Quinn et al., 1985) as basic medium, which was served as control. The centrifugation for removing seminal plasma before dilution with cryoprotective media and subsequent cryopreservation at 700 g for 6 min was performed. This allowed us to achieve very high (~ 80%) amount of spermatozoa

(Figure 9). Integral membrane proteins are associated with the lipid bilayer and their function may be expected to be altered, especially those that perform the function of transport channels for calcium absorption. The permeability of these channels is increased on cooling, affecting calcium regulation (Robertson & Watson, 1986; Robertson et al., 1988). These facts have serious consequences for cell function (Bailey & Bhur, 1994) and many changes may be incompatible with sperm viability. In this case we have decided to apply to dog spermatozoa the early developed us vitrification protocol (Isachenko et al., 2008) for human sperm cells. The following tested groups were compared: HTF (Control); HTF– bovine serum albumin (BSA, 1% end-concentration); HTF–BSA + 0.1 M sucrose; HTF–BSA +

The vitrification procedure was done as follow. Briefly, aliquots of 30 µl of sperm suspension (different vitrification media) were dropped directly into LN2. After solidification, the spheres were packaged in cryotubes and stored for at least 24 h in liquid nitrogen before use. The warming was performed by quickly submerging spheres one by one (not more than five spheres) in 5 ml of HTF–BSA 1% pre-warmed to 37°C accompanied by gentle agitation for 5–10 sec. The post-thaw sperm suspension was maintained at 37°C and 5% CO2 for 10 min and then centrifuged at 300 g for 5 min. The cell pellet was finally re-

The influence of tested media on the following physiological parameters of dog spermatozoa we have checked with such screening methods: viability and condition of acrosome with double stain technique (Trypan blue–Giemsa) with subsequent evaluation of acrosome pattern according to Didion (Didion et al., 1989); DNA fragmentation was detected with using of TUNEL technique (Gorczyca et al., 1993); detection of the change in mitochondrial permeability was done according to Smiley (Smiley et al., 1991); the motility

According to our investigtion the percentage of spermatozoa with acrosome-intact membrane was high in all treatment groups (Figure 9) independent from concentration of

The best progressive motility after warming (Figure 10) was significantly increased in the sperm vitrified with 0.25 M sucrose and 1% BSA (42.5 ± 2.3%), compared to other treatment groups (P < 0.01). However, lower or higher concentration of sucrose did not significantly improve the progressive motility post-vitrification. Comparable results (60.7% of motility) was reported (Tsutsui et al., 2003) when the dog semen was chilled in egg yolk–\*\*Tris at 4°C

The presence of sucrose in vitrification solution independent from the concentration has strong positive influence on viability of spermatozoa (Figure 10) and was ~70% (P<0.001) for

with intact acrosome in control.

0.25 M sucrose and HTF–BSA + 0.4 M sucrose.

suspended in 50 µl of HTF only for sperm evaluation.

sucrose in vitrification solution, but lower then in control (P<0.05).

for over 4 days, but the spermatozoa lost their fertilizing capacity.

of spermatozoa was checked as well.

all sucrose-treatment groups.
