**4. Technology for vitrification of dog spermatozoa (Sánchez et al, 2011)**

If we will investigate the history of reproductive cryobiology we will see that all routine-used technique, excluding intracytoplasmic sperm injection, was firstly approved on the animal model. The same happened and with vitrification technique. After first promising investigation with frog (Luyet and Hodapp, 1938), human (Jahnel, 1938; Parkes, 1945) fowl (Schaffner, 1942) and human, and rabbit (Hoagland and Pincus1942) spermatozoa the vitrification technique was successful re-discovered in 2002 (Nawroth et al., 2002) on human spermatozoa. Recently we have decided to extrapolate the results of our investigation on animal model, thus we have with high attention examined the work of Watson and Plummer (Watson and Plummer, 1985) about responses of spermatozoa from different kind of animals to cold shock. According to this work most sensitive to cold injury are spermatozoa of animals, which produce gametes with big blade-shaped flat head. The spermatozoa of human, stallion, dog and cat have the highest stability to cold shock due to smallest blade-shaped flat head compare to the rabbit spermatozoa (have middle stability), ram, bull and boar (have the lowest stability to cold shock). Took into account these data we have decided, that the spermatozoa from human, stallion, dog and cat could be similar well preserved using vitrification technique. In this case we have decided to investigate the ability of dog spermatozoa, which stay on third place after human one according to head's size, to maintain their physiological function after vitrification without use of permeable cryoprotectants. In cryobiological routine practice, carbohydrates were already used for sperm cryopreservation (Nakagata, Takeshima, 1992, 1993; Wakayama et al., 1998). It has been suggested that raffinose plays the role of a membrane stabilizing and dehydrating agent. Comparative investigation of three different sugars, monosaccharide glucose, disaccharide sucrose and trisaccharide raffinose, showed that protection against freezing/thawing injuries is independent of the kind of sugar itself, but depends more on the sugar's concentration (Koshimoto & Mazur, 2002). Based on this evidence, we have decided to investigate the different concentrations of sucrose on the viability of cryopreserved spermatozoa. However, the problem was that the dog spermatozoa have the special physiological property which connect to capacitation process. It is well known that spermatozoa of different kind mammalian species are very sensitive to the negative effect of cryoprotectants dependent on temperature (Sánchez & Schill, 1991; Deppe et al., 2004). It is also proved (Pérez et al., 1996) that low survival and fertilizing capacity of cryopreserved mammalian spermatozoa has been attributed to an early state of capacitation resulting from the procedures by which spermatozoa are preserved. These kind of changes have been called as *'early state capacitation'* or '*cryocapacitation'.* These data of Pérez and colleagues (Pérez et al., 1996) later were supported (Samper, 1997; Maxwell et al., 1997). These authors showed the negative effect of cryocapacitation on the fertilizing capacity and viability of spermatozoa. The described study showed that the canine spermatozoa starts rapidly with capacitation process as soon as have been separated from seminal plasma with subsequent 20–40% of capacitated and spontaneous acrosome reacted spermatozoa in culture media. This rate is higher than in other mammals (Risopatrón et al., 2002; Santiani et al., 2004) and the spermatozoa are therefore more affected by the cryocapacitation process than those of other species. Probably the sperm membrane in this species is especially sensitive to cooling in the range of temperatures between 20°C and 5°C and to heating to 30°C at thawing (Holt & North, 1991; Sánchez & Schill, 1991). In this case for dog spermatozoa which are high sensitive to capacitation the cryopreservation protocol with a very increased cooling speed should be used, because these temperature ranges by ultra-rapid freezing (vitrification) will be just eliminated.

If we will investigate the history of reproductive cryobiology we will see that all routine-used technique, excluding intracytoplasmic sperm injection, was firstly approved on the animal model. The same happened and with vitrification technique. After first promising investigation with frog (Luyet and Hodapp, 1938), human (Jahnel, 1938; Parkes, 1945) fowl (Schaffner, 1942) and human, and rabbit (Hoagland and Pincus1942) spermatozoa the vitrification technique was successful re-discovered in 2002 (Nawroth et al., 2002) on human spermatozoa. Recently we have decided to extrapolate the results of our investigation on animal model, thus we have with high attention examined the work of Watson and Plummer (Watson and Plummer, 1985) about responses of spermatozoa from different kind of animals to cold shock. According to this work most sensitive to cold injury are spermatozoa of animals, which produce gametes with big blade-shaped flat head. The spermatozoa of human, stallion, dog and cat have the highest stability to cold shock due to smallest blade-shaped flat head compare to the rabbit spermatozoa (have middle stability), ram, bull and boar (have the lowest stability to cold shock). Took into account these data we have decided, that the spermatozoa from human, stallion, dog and cat could be similar well preserved using vitrification technique. In this case we have decided to investigate the ability of dog spermatozoa, which stay on third place after human one according to head's size, to maintain their physiological function after vitrification without use of permeable cryoprotectants. In cryobiological routine practice, carbohydrates were already used for sperm cryopreservation (Nakagata, Takeshima, 1992, 1993; Wakayama et al., 1998). It has been suggested that raffinose plays the role of a membrane stabilizing and dehydrating agent. Comparative investigation of three different sugars, monosaccharide glucose, disaccharide sucrose and trisaccharide raffinose, showed that protection against freezing/thawing injuries is independent of the kind of sugar itself, but depends more on the sugar's concentration (Koshimoto & Mazur, 2002). Based on this evidence, we have decided to investigate the different concentrations of sucrose on the viability of cryopreserved spermatozoa. However, the problem was that the dog spermatozoa have the special physiological property which connect to capacitation process. It is well known that spermatozoa of different kind mammalian species are very sensitive to the negative effect of cryoprotectants dependent on temperature (Sánchez & Schill, 1991; Deppe et al., 2004). It is also proved (Pérez et al., 1996) that low survival and fertilizing capacity of cryopreserved mammalian spermatozoa has been attributed to an early state of capacitation resulting from the procedures by which spermatozoa are preserved. These kind of changes have been called as *'early state capacitation'* or '*cryocapacitation'.* These data of Pérez and colleagues (Pérez et al., 1996) later were supported (Samper, 1997; Maxwell et al., 1997). These authors showed the negative effect of cryocapacitation on the fertilizing capacity and viability of spermatozoa. The described study showed that the canine spermatozoa starts rapidly with capacitation process as soon as have been separated from seminal plasma with subsequent 20–40% of capacitated and spontaneous acrosome reacted spermatozoa in culture media. This rate is higher than in other mammals (Risopatrón et al., 2002; Santiani et al., 2004) and the spermatozoa are therefore more affected by the cryocapacitation process than those of other species. Probably the sperm membrane in this species is especially sensitive to cooling in the range of temperatures between 20°C and 5°C and to heating to 30°C at thawing (Holt & North, 1991; Sánchez & Schill, 1991). In this case for dog spermatozoa which are high sensitive to capacitation the cryopreservation protocol with a very increased cooling speed should be used, because these

**4. Technology for vitrification of dog spermatozoa (Sánchez et al, 2011)** 

temperature ranges by ultra-rapid freezing (vitrification) will be just eliminated.

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 with intact acrosome in control.

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

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 resuspended in 50 µl of HTF only for sperm evaluation.

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 of spermatozoa was checked as well.

According to our investigtion the percentage of spermatozoa with acrosome-intact membrane was high in all treatment groups (Figure 9) independent from concentration of sucrose in vitrification solution, but lower then in control (P<0.05).

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

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 all sucrose-treatment groups.

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 61

well as higher concentrations (0.4 M) of sucrose had not significantly protective effect against DNA fragmentation. These data support our previous results (Isachenko et al., 2004a, b, the vitrification medium included only 1% HSA) and we can assume that vitrification itself due to very fast speed of cooling can provide protective effect on DNA and protect against fragmentation. It is very important results, because damage of DNA in sperm is strongly correlated with mutagenic events (Moreno et al., 2004) and how have showed Paasch with colleagues (Paasch et al., 2004) cryopreservation and thawing can be associated with varying extent of activation of apoptotic machinery in human spermatozoa. The danger is that such spermatozoa are still able to fertilize the oocyte, however, the mutations and defects did not possible to discover until the embryo has divided and the fetus has developed (Twigg et al., 1998). At present exist the opinion that DNA decondensation or fragmentation may occur in different magnitudes, which will depend on the process or the kind of cryoprotectant used (Schuffner et al., 2001; Chohan et al., 2004; Ngamwuttiwong & Kunathikom, 2007; Yildiz et al., 2007). Unfortunately, until now this question is still open, because it is not entirely clear what the effect of cryopreservation on DNA integrity is, and what would be the ideal conditions of slow freezing to reduce this effect. In this case the method of vitrification is more successful, because allows to obtain low levels of DNA fragmentation by protection due to applying of very high speed of

cooling and exclusion of permeable cryoprotectants from vitrification solution.

Fig. 11. DNA fragmentation in canine sperm after vitrification with 1%BSA and different concentrations of sucrose. DNA fragmentation was determined by the TUNEL assay. Data are expressed as mean ± SD from six experiments. A significant difference with respect to the control is indicated by a (P < 0.05). Control: Sperm vitrified with medium HTF only.

Fragmentation DNA has been interpreted at present as apoptosis or apoptosis-like events (Paasch et al., 2004). This has been verified in cryopreserved / thawed sperm, with presence of increased caspase's activity induced by cryopreservation (caspase-3, -8, -9), decreased M due to release of regulating proteins associated with mitochondria, evidence of DNA

BSA, bovine serum albumin.

Fig. 9. Acrosome intact in canine spermatozoa after vitrification with 1% BSA and different concentrations of sucrose. Percentage of acrosome intact spermatozoa was determined by dual stain (Trypan blue– Giemsa). Data are expressed as mean ± SD from six experiments. Control = Sperm vitrified with medium HTF only. BSA, bovine serum albumin.

Fig. 10. Progressive motility and viability of canine spermatozoa after vitrification with 1% BSA and different concentrations of sucrose. Motility was determined by microscopic examination using a phase contrast microscope and viability by dual stain (Trypan blue– Giemsa). Data are expressed as mean ± SD from six experiments. A significant difference with respect to the control is indicated by a (P < 0.01). Control: Sperm vitrified with medium HTF only. BSA, bovine serum albumin.

Our data have shown that the vitrification significantly protect the sperm DNA (Figure 11) against fragmentation when used 0.25 M sucrose in combination with 1% BSA compare to control (97,2 ± 0.5% vs 94,4 ± 0.6%, respectively, P < 0.05). However, the lower (0.1 M) as

Fig. 9. Acrosome intact in canine spermatozoa after vitrification with 1% BSA and different concentrations of sucrose. Percentage of acrosome intact spermatozoa was determined by dual stain (Trypan blue– Giemsa). Data are expressed as mean ± SD from six experiments. Control = Sperm vitrified with medium HTF only. BSA, bovine serum

Fig. 10. Progressive motility and viability of canine spermatozoa after vitrification with 1% BSA and different concentrations of sucrose. Motility was determined by microscopic examination using a phase contrast microscope and viability by dual stain (Trypan blue– Giemsa). Data are expressed as mean ± SD from six experiments. A significant difference with respect to the control is indicated by a (P < 0.01). Control: Sperm vitrified with medium

Our data have shown that the vitrification significantly protect the sperm DNA (Figure 11) against fragmentation when used 0.25 M sucrose in combination with 1% BSA compare to control (97,2 ± 0.5% vs 94,4 ± 0.6%, respectively, P < 0.05). However, the lower (0.1 M) as

albumin.

HTF only. BSA, bovine serum albumin.

well as higher concentrations (0.4 M) of sucrose had not significantly protective effect against DNA fragmentation. These data support our previous results (Isachenko et al., 2004a, b, the vitrification medium included only 1% HSA) and we can assume that vitrification itself due to very fast speed of cooling can provide protective effect on DNA and protect against fragmentation. It is very important results, because damage of DNA in sperm is strongly correlated with mutagenic events (Moreno et al., 2004) and how have showed Paasch with colleagues (Paasch et al., 2004) cryopreservation and thawing can be associated with varying extent of activation of apoptotic machinery in human spermatozoa. The danger is that such spermatozoa are still able to fertilize the oocyte, however, the mutations and defects did not possible to discover until the embryo has divided and the fetus has developed (Twigg et al., 1998). At present exist the opinion that DNA decondensation or fragmentation may occur in different magnitudes, which will depend on the process or the kind of cryoprotectant used (Schuffner et al., 2001; Chohan et al., 2004; Ngamwuttiwong & Kunathikom, 2007; Yildiz et al., 2007). Unfortunately, until now this question is still open, because it is not entirely clear what the effect of cryopreservation on DNA integrity is, and what would be the ideal conditions of slow freezing to reduce this effect. In this case the method of vitrification is more successful, because allows to obtain low levels of DNA fragmentation by protection due to applying of very high speed of cooling and exclusion of permeable cryoprotectants from vitrification solution.

Fig. 11. DNA fragmentation in canine sperm after vitrification with 1%BSA and different concentrations of sucrose. DNA fragmentation was determined by the TUNEL assay. Data are expressed as mean ± SD from six experiments. A significant difference with respect to the control is indicated by a (P < 0.05). Control: Sperm vitrified with medium HTF only. BSA, bovine serum albumin.

Fragmentation DNA has been interpreted at present as apoptosis or apoptosis-like events (Paasch et al., 2004). This has been verified in cryopreserved / thawed sperm, with presence of increased caspase's activity induced by cryopreservation (caspase-3, -8, -9), decreased M due to release of regulating proteins associated with mitochondria, evidence of DNA

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 63

instead of cylindrical; thus, the flagellum appears brighter by dark-field microscopy, allowing clear visualization of wave shapes (Cosson et al. 2008). Just for knowledge, the head of investigated us rainbow trout spermatozoa is ovoid-shaped, measuring about 3 x 1.3 µm in diameter and possess any acrosome. In middle piece present only one mitochondrial body (several mitochondria are sometimes identified in the middle piece, but later during evolution they are fused together) is shaped like an incompletely closed ring. The middle piece is completely separated from the flagellum by an invagination of the cell membrane, which reaches from the head to the base (Billard, 1983; Tuset et al., 2008). During spermatogenesis, sperm cells are prepared for accomplishing their fertilizing task for which they need to fully exploit their swimming ability immediately and as fast as possible in order to encounter the egg. The initial velocity is very high at activation, but motility duration lasts for periods ranging only 40 s to 20 min as an energetic consequence of the high velocity (Cosson et al. 2008). As possible to see the fish spermatozoa are much different

Since the first successful cryopreservation of herring sperm 50 years ago (Blaxter, 1953) considerable improvement has been achieved in sperm cryopreservation and developed technology of conventional freezing of fish spermatozoa has been used in agricultural practice very broadly (Scott and Baynes, 1980; Stoss and Holtz, 1981; Dreanno et al., 1997; Wheeler and Thorgaard, 1991; Conget et al., 1996; Lahnsteiner et at., 2000; Fabbrocini et al., 2000; Zhang et al., 2003; Chen et al., 2004; Viveiros and Godinho, 2009). Usually, for protection of spermatozoa from the negative effects of low temperatures caused by conventional freezing ('slow', with controlled rate of cooling), permeable cryoprotectants are used. At present, applied cryobiology practically always uses only four permeable cryoprotectants: three spirits (ethylene glycol, propylene glycol and glycerol) and the highly polarized organic solvent dimethyl sulfoxide. However, as reported for mammalian spermatozoa, these cryoprotectants can produce osmotic and cytotoxic effects, including parthenogenesis (Gilmore et al., 1997). And for fish spermatozoa these problems are still very actual, because post-thaw viability and fertility of the cryopreserved sperm are reduced dramatically as a result of accumulated cellular damage that arise throughout the freezingthawing process. The same like for other species the cryopreservation results in considerable damage to cellular structures such as plasma membrane, nucleus, mitochondria, and flagellum (Lahnsteiner et al., 1992; 1996; Drokin et al., 1998; Conget et al., 1996; Zhang et al., 2003). So, according to Ogier de Baulny (Ogier de Baulny, 1997) the percentage of spermatozoa with an intact membrane and a functional mitochondrion after cryopreservation varied below 18% only. According to our results which we have achieved on human spermatozoa (Isachenko et al. 2003, 2004a,b, 2005, 2008, 2011a, b, c, d) and dog (Sánchez et al., 2011b) with applying of cryoprotectant-free vitrification protocol we have decided to investigate the method on fish spermatozoa (*Oncorhynchus mykiss*) (Merino et al., 2011a, b). This decision we have got because the authors of these studies were able to establish statistically higher motility and in vitro fertilization ability of vitrified spermatozoa

compared with spermatozoa cryopreserved using conventional slow freezing.

The standard Cortland® culture medium (Trus-Cott et al., 1968) for fish spermatozoa (per liter: 1.88 g NaCl, 0.23 g CaCl2, 7.2 g KCl, 0.41 g NaH2PO4, 1 g NaHCO3, 0.23 g MgSO4·7 H2O, 1.0 g Glucose, 10% Glycol and 10% Tris Base and prepared to pH 8 at 268mOsm) was used for all manipulation and served as control. Fresh-retrieved semen was diluted 1:3 in the non-activating Cortland® medium with subsequent determination of the motility and concentration by phase-

from mammalian one.

fragmentation, externalization of phosphatidylserine in the plasma membrane (Paasch et al., 2004) and production of reactive oxygen substances (Roca et al., 2005). Among the first events that occur in early apoptosis are changes in mitochondrial permeability which alter the transmembrane potential (M ). Changes in the M are caused by the insertion of proapoptotic proteins within the membrane, and oligomerisation may create pores, dissipating the transmembrane potential and thus releasing cytochrome c into the cytoplasm (Zamzami et al., 1995). In our work (Figure 12) we achieved the reduction of apoptotic-like process in canine spermatozoa and have got after warming more then 40% of spermatozoa with intact M using of the vitrification solution with 0.25 M sucrose and 1% BSA compare to other treatment groups (P<0.001).

These results have demonstrated that vitrification without the use of permeable cryoprotectants allows to avoid the cryoprotectants toxicity caused by their addition and removal with subsequent negative effects on the spermatozoa genome. The use of sucrose in concentration of 0.25 M in combination with 1% BSA and ultrarapid speed of cooling can effectively preserve important physiological parameters of canine spermatozoa.

Fig. 12. Integrity of mitochondrial membrane potential in canine spermatozoa after vitrification with 1% BSA and different concentrations of sucrose. Mitochondrial membrane potential was determined by staining with the cationic fluorescent JC-1. Data are expressed as mean ± SD from six experiments. A significant difference with respect to the control is indicated by a (P < 0.05) and b (P < 0.01). Control: Sperm vitrified with medium HTF only. BSA, bovine serum albumin.
