**5. New technology for vitrification of fish (Oncorhynchus mykiss) spermatozoa Merino et al., 2011a, b.**

At the beginning of this sub-chapter we would like to mention that the fish spermatozoa of both sea and river fish species have a very special peculiarities compare to all mammalian species. The fish sperm cells are homogenous; all spermatozoa can be activated at the same time and then swim with very similar characteristics at a certain time point post-activation. In many fish species, the flagellum is 50–60 mm long with a ribbon shape (presence of fins)

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

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

**5. New technology for vitrification of fish (Oncorhynchus mykiss)** 

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.

At the beginning of this sub-chapter we would like to mention that the fish spermatozoa of both sea and river fish species have a very special peculiarities compare to all mammalian species. The fish sperm cells are homogenous; all spermatozoa can be activated at the same time and then swim with very similar characteristics at a certain time point post-activation. In many fish species, the flagellum is 50–60 mm long with a ribbon shape (presence of fins)

compare to other treatment groups (P<0.001).

BSA, bovine serum albumin.

**spermatozoa Merino et al., 2011a, b.** 

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 from mammalian one.

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-

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 65

Sperm samples were centrifuged at 300 g for 10 min at 4 ◦C. The seminal plasma (supernatant) was retained and the sperm suspension diluted with Cortland® medium to a concentration of 40×106 spermatozoa/ml. Five equal 500-µl aliquots from each preparation were placed in individual 1ml tubes for vitrification. Twenty microliters of sperm suspension from each tube was dropped directly into liquid nitrogen, during which the droplet adopted a spherical form approximately 3mm in diameter. After 5min, the solidified droplets were placed into 2-ml cryovials pre-cooled in liquid nitrogen with precooled tweezers. After storage for at least 24 h in liquid nitrogen, the samples were warmed by plunging the droplets into a 15ml tube containing 5ml Cortland® medium supplemented with 1% BSA at 37°C with intense agitation. After warming (one droplet/tube), the tubes

In our work we have investigated the following five treatments groups (Figure 13):

**Group 5:** Cortland® medium+ 1% BSA + 40% seminal plasma + 0.125M sucrose. The vitrification /warming of rainbow spermatozoa was proceeded as following:

were maintained at 37°C for 5–10 min prior to evaluation of spermatozoa quality.



To investigate these cold sensitive (Holt, 2000; O'Connell et al., 2002) organelles of spermatozoa were necessary because the retention of plasma membrane integrity and

The spermatozoa quality was tested according the following parameters:

which is why staining is done after inhibitor treatment (Figure 15).

drb KT 450905, Zeiss) at 400x magnification (Figure 14).

**Group 1:** Cortland® medium only (frozen control)

**Group 3:** Cortland® medium+ 1% BSA + 0.125M sucrose

**Group 4**: Cortland® medium+ 1% BSA + 40% seminal plasma

**Group 2**: Cortland® medium+ 1% BSA

contrast microscopy. Subjective evaluations of motility were performed by placing 2µl of this sperm suspension on a glass slide and immediately adding 10µl of the activator Powermilt® (Católica of Temuco University, Chile) at 10°C. The motility of the spermatozoa was observed in 12µl sperm activated by subjective microscopic examination under phase contrast optics at 400x magnification. Motility assessments were made in triplicate for each sample at 5 s following activation with Powermilt®. Sperm concentrations were determined with a Neubauer hemocytometer after dilution of 1µl of sperm suspension in 1200µl of standard culture medium. Only samples with high motility (>80%) and concentration 12×109 spermatozoa/mL (Drokin et al., 1998) were used in this study.

Fig. 13. Motility, cytoplasmic membrane integrity and mitochondrial membrane integrity of vitrified rainbow trout spermatozoa. (CM) Cortland®, (BSA) 1% bovine serum albumin, (SP) 40% of seminal plasma, (S) 0.125 M sucrose. Different superscripts indicate statistical difference between respective values of compared groups (P<0.05).

In our work we have investigated the following five treatments groups (Figure 13):

**Group 1:** Cortland® medium only (frozen control)

**Group 2**: Cortland® medium+ 1% BSA

64 Current Frontiers in Cryobiology

contrast microscopy. Subjective evaluations of motility were performed by placing 2µl of this sperm suspension on a glass slide and immediately adding 10µl of the activator Powermilt® (Católica of Temuco University, Chile) at 10°C. The motility of the spermatozoa was observed in 12µl sperm activated by subjective microscopic examination under phase contrast optics at 400x magnification. Motility assessments were made in triplicate for each sample at 5 s following activation with Powermilt®. Sperm concentrations were determined with a Neubauer hemocytometer after dilution of 1µl of sperm suspension in 1200µl of standard culture medium. Only samples with high motility (>80%) and concentration 12×109 spermatozoa/mL (Drokin et

Fig. 13. Motility, cytoplasmic membrane integrity and mitochondrial membrane integrity of vitrified rainbow trout spermatozoa. (CM) Cortland®, (BSA) 1% bovine serum albumin, (SP) 40% of seminal plasma, (S) 0.125 M sucrose. Different superscripts indicate statistical

difference between respective values of compared groups (P<0.05).

al., 1998) were used in this study.

**Group 3:** Cortland® medium+ 1% BSA + 0.125M sucrose

**Group 4**: Cortland® medium+ 1% BSA + 40% seminal plasma

**Group 5:** Cortland® medium+ 1% BSA + 40% seminal plasma + 0.125M sucrose.

The vitrification /warming of rainbow spermatozoa was proceeded as following:

Sperm samples were centrifuged at 300 g for 10 min at 4 ◦C. The seminal plasma (supernatant) was retained and the sperm suspension diluted with Cortland® medium to a concentration of 40×106 spermatozoa/ml. Five equal 500-µl aliquots from each preparation were placed in individual 1ml tubes for vitrification. Twenty microliters of sperm suspension from each tube was dropped directly into liquid nitrogen, during which the droplet adopted a spherical form approximately 3mm in diameter. After 5min, the solidified droplets were placed into 2-ml cryovials pre-cooled in liquid nitrogen with precooled tweezers. After storage for at least 24 h in liquid nitrogen, the samples were warmed by plunging the droplets into a 15ml tube containing 5ml Cortland® medium supplemented with 1% BSA at 37°C with intense agitation. After warming (one droplet/tube), the tubes were maintained at 37°C for 5–10 min prior to evaluation of spermatozoa quality.

The spermatozoa quality was tested according the following parameters:


To investigate these cold sensitive (Holt, 2000; O'Connell et al., 2002) organelles of spermatozoa were necessary because the retention of plasma membrane integrity and

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 67

Fig. 15. Example of rainbow trout spermatozoa with (A) non-damaged and (B) damaged

treated spermatozoa (5.5%, 49.8%, 37.1%, 54.7%, and 34.4%, respectively). As possible to see from our results they can have the following potential question. How spermatozoa can have a high level of motility with low level of the integrity of mitochondrial membranes? Especially this question is actual taking into account that fish spermatozoa have only one mitochondrion. A lower M , as reflected by green fluorescence, simply can have the following explanation. Activity of the mitochondria, including ATP production, is reduced when compared to their red counterparts. Cell often have "green" and "red" mitochondria with shifts between all green or all red governed by a variety of external and internal conditions, and this is normal. Individual mitochondria constantly shift from red to green and back to red in response to rapid changes in local conditions, including calcium levels and pH (Vanblerkom, personal communication). While the plasma membrane is known to be sensitive to cryopreservation (Cabrita et al., 2001; Aitken and Baker, 2006; Muller et al., 2008), our results shows that it is cryostable in rainbow trout sperm, as indicated by ~90% of non-damaged plasmatic membrane in sperm vitrified in culture medium only i.e., without permeable cryoprotectants and additional proteins). This is similar to levels reported after conventional freezing with permeable cryoprotectants. We suggest that the vitrification technique described here which associated with high rate of cooling allows to avoid the formation of large extracellular water crystals. Sucrose is well known to have a beneficial influence on the plasma membrane of cells subjected to cryopreservation (Anchordoguy et al., 1987; Rodgers and Glaser, 1993). For human spermatozoa, the drop-wise technique of vitrification is a major technical advance because it includes a mixture of non-permeable cryoprotectants such as serum albumin (Isachenko et al., 2008). However, we report that the

mitochondria. Bar = 2.5µm.

mitochondrial function after cryopreservation is too important with regard to fertilization capacity of both spermatozoa and oocytes (Gao et al., 1997; de Lamirande et al., 1997). For all species, normal mitochondrial function is a key factor in the fertilizability of spermatozoa and for fish it is especially critical to maintain mitochondrial activity because high motility normally lasts for only 30 s to few minutes. Spermatozoa of rainbow trout have only one mitochondrion to produce sufficient ATP to drive this transient high motility, and damage during cryopreservation will certainly lead to decrease of motility and as a result, fertilization ability (Maisse, 1996). In this case the stability of mitochondrion during cryopreservation can be used as a specific test for applicability of a any investigated cryopreservation protocol (Meseguer et al., 2004; O'Connell et al., 2002).

The results of this investigation showed that the proportion of sperm showing normal, high motility varied between 82% and 95% in fresh samples. In Groups 1, 2, 3, 4, and 5, motility in these solutions was 86%, 71%, 79%, 81%, and 82%, respectively (Figure 13).

The percent of spermatozoa with intact cytoplasmic membrane after thawing was similar between the 5 experimental groups, ranging from 81.8% to 90%, as shown in Figures 13 and 14. Nevertheless, the integrity of mitochondrial membrane potential of spermatozoa (Figures 13 and 15) in Groups 1, 2, 3, 4, and 5 was decreased significantly compare to non

Fig. 14. Example of rainbow trout spermatozoa with non-damaged (green) and damaged (red) cytoplasmic membranes. Bar = 8µm.

mitochondrial function after cryopreservation is too important with regard to fertilization capacity of both spermatozoa and oocytes (Gao et al., 1997; de Lamirande et al., 1997). For all species, normal mitochondrial function is a key factor in the fertilizability of spermatozoa and for fish it is especially critical to maintain mitochondrial activity because high motility normally lasts for only 30 s to few minutes. Spermatozoa of rainbow trout have only one mitochondrion to produce sufficient ATP to drive this transient high motility, and damage during cryopreservation will certainly lead to decrease of motility and as a result, fertilization ability (Maisse, 1996). In this case the stability of mitochondrion during cryopreservation can be used as a specific test for applicability of a any investigated

The results of this investigation showed that the proportion of sperm showing normal, high motility varied between 82% and 95% in fresh samples. In Groups 1, 2, 3, 4, and 5, motility

The percent of spermatozoa with intact cytoplasmic membrane after thawing was similar between the 5 experimental groups, ranging from 81.8% to 90%, as shown in Figures 13 and 14. Nevertheless, the integrity of mitochondrial membrane potential of spermatozoa (Figures 13 and 15) in Groups 1, 2, 3, 4, and 5 was decreased significantly compare to non

Fig. 14. Example of rainbow trout spermatozoa with non-damaged (green) and damaged

(red) cytoplasmic membranes. Bar = 8µm.

cryopreservation protocol (Meseguer et al., 2004; O'Connell et al., 2002).

in these solutions was 86%, 71%, 79%, 81%, and 82%, respectively (Figure 13).

Fig. 15. Example of rainbow trout spermatozoa with (A) non-damaged and (B) damaged mitochondria. Bar = 2.5µm.

treated spermatozoa (5.5%, 49.8%, 37.1%, 54.7%, and 34.4%, respectively). As possible to see from our results they can have the following potential question. How spermatozoa can have a high level of motility with low level of the integrity of mitochondrial membranes? Especially this question is actual taking into account that fish spermatozoa have only one mitochondrion. A lower M , as reflected by green fluorescence, simply can have the following explanation. Activity of the mitochondria, including ATP production, is reduced when compared to their red counterparts. Cell often have "green" and "red" mitochondria with shifts between all green or all red governed by a variety of external and internal conditions, and this is normal. Individual mitochondria constantly shift from red to green and back to red in response to rapid changes in local conditions, including calcium levels and pH (Vanblerkom, personal communication). While the plasma membrane is known to be sensitive to cryopreservation (Cabrita et al., 2001; Aitken and Baker, 2006; Muller et al., 2008), our results shows that it is cryostable in rainbow trout sperm, as indicated by ~90% of non-damaged plasmatic membrane in sperm vitrified in culture medium only i.e., without permeable cryoprotectants and additional proteins). This is similar to levels reported after conventional freezing with permeable cryoprotectants. We suggest that the vitrification technique described here which associated with high rate of cooling allows to avoid the formation of large extracellular water crystals. Sucrose is well known to have a beneficial influence on the plasma membrane of cells subjected to cryopreservation (Anchordoguy et al., 1987; Rodgers and Glaser, 1993). For human spermatozoa, the drop-wise technique of vitrification is a major technical advance because it includes a mixture of non-permeable cryoprotectants such as serum albumin (Isachenko et al., 2008). However, we report that the

Vitrification Technique – New Possibilities for Male Gamete Low-Temperature Storage 69

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inclusion of sucrose in the vitrification solution was ineffective for rainbow trout spermatozoa. According to Lahnsteiner (2007), lipoproteins in the seminal plasma of rainbow trout likely maintain the lipid composition of the plasma and may increase the cryostability of spermatozoa. Our results support this point of view and we suggest that the method of sperm vitrification described here could also be applied to other species. As a rule, carbohydrates are used for sperm cryopreservation to compensate for the decrease in osmotic pressure caused by the permeable cryoprotectant glycerol, which works as an additional dissolvent and has the ability to decrease the medium's osmotic pressure. Based on this evidence, we investigated whether sucrose had a similar cryoprotective effect on fish spermatozoa during freeze–thaw. We found that its inclusion in vitrification medium has no visible protective effect on mitochondrial membrane integrity nor does it provide significant protection for spermatozoa when compared to other vitrification mediums containing BSA or BSA + seminal plasma. Indeed, the addition of these non-permeable cryoprotectants did not increase either the motility or plasma membrane integrity of rainbow trout spermatozoa. However, described here technology of cryopreservation of fish spermatozoa by direct plunging into liquid nitrogen has big disadvantage because did not protect the biological material against direct contact with liquid nitrogen. In this connection in the future investigation it would be necessary to find a synthetic substitute for seminal plasma to avoid the possible microbial contamination. In fact, any technology in reproductive biology, and especially in a therapeutic medical approach, must guarantee the full protection of cells from microorganisms that might survive in liquid nitrogen temperatures (Gardner, 1998; Bielanski et al., 2003), and it has been suggested that liquid nitrogen can be contaminated by microorganisms (Tedder et al., 1995). The problem of potential microbial contamination of spermatozoa during cryopreservation, especially by the virus of Infectious Salmon Anemia is significant in the fish industry, especially in Latin America (Ellis, 2007; Fortt and Buschmann, 2007; Sommer, 2009). In spite of that the results of our experiments conformed that for fish spermatozoa the developed method of cryopreservation by direct plunging into liquid nitrogen (vitrification) without permeable cryoprotectants is potentially significant for this industry, but the development of "aseptic" methods, in which the spermatozoa suspension is enclosed in capillaries or straws to prevent direct contact of sperm with liquid nitrogen, will need to be considered. Filtration or ultraviolet treatment of liquid nitrogen cannot guarantee the absence of contamination of biological material by viruses. For example, Tedder et al. (1995) reported the contamination of blood probes by hepatitis virus during the storage of probes in liquid nitrogen. Different types of viruses, such as hepatitis virus, papova virus, vesicular stomatitis virus and herpes virus, which are simple and very cryostable structures, may increase their virulence after direct plunging and storage in liquid nitrogen (Hawkins et al., 1996; Charles and Sire, 1971; Schaffer et al., 1976; Jones and Darville, 1989).
