**7. Kinetic vitrification of sperm: why some species have while others have not been vitrified?**

Now, as we are fully equipped to discuss the core topic of the Chapter, let us refresh the turn of (relatively) recent events related to the *kinetic* VF of spermatozoa.

## **7.1 A turn of the helix: The Isachenkos' experiments on vitrification of human sperm**

As we mentioned in the Introductory sub-chapter, 1, after earlier attempts to vitrify sperm with contradictory results, the findings of the cryoprotective role of glycerol and other CPAs at slow freezing moved the field of cryopreservation of spermatozoa from early attempts of K-VF toward E-SF. It has been successfully applied to many types of sperm, yet somewhere in 1990s, the data started accumulating that suggested that glycerol, DMSO and other permeable CPAs might adversely affect the genetic and especially epigenetic fabric of spermatozoa. At the same time, several Japanese groups had successful CP of very sensitive mouse spermatozoa without any permeable CPA but with 18% of impermeable raffinose (a 3-ring sugar) and a mixture of proteins (skim milk) [Okuyama *et al.*, 1990; Tada *et al.*, 1990; Yokoyama *et al.*, 1990]. It worked so exceptionally well, that the Mazur's group, which had originally cryopreserved mouse sperm with glycerol [Mazur *et al.*, 2000] (though found that it can be indeed chemically toxic to the sperm [Katkov *et al.*, 1998]) finally also reported that fast immersion of mouse spermatozoa into liquid nitrogen without any CPA worked perfectly [Koshimoto *et al.*, 2000]. In any case, those data had inspired Evgenia and Vladimir Isachenko to freeze human sperm in tiny pellicles by plunging those "cryogenic loops" without any CPA whatsoever. They published their findings in 2002, and a year later, the explanation why it worked was followed [Isachenko *et al.*, 2003; Nawroth *et al.*, 2002]. That marked the *"second wind"* in the kinetic VF of spermatozoa. The history of the development is described in numerous papers [Isachenko *et al.*, 2004a; Isachenko *et al.*, 2008; Isachenko *et al.*, 2004b; Isachenko *et al.*, 2005] and several reviews by the authors [Isachenko *et al.*, 2007; Isachenko *et al.*, 2010; Katkov *et al.*, 2007] and briefly touched in this Book in Chapter 2 [Isachenko *et al.*, 2012]. The method has been involved from a cryo loop (pellicle) through droplets in LN2 to quite elaborated "aseptic technology". Some of the carriers used by the Isachenkos at different stages are shown in **Fig. 6**.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 25

Table 1. Comparison of statements published by Isachenkos et al in 2003-4 and by other

*thoroughly investigated"* makes sense in the matter of describing a particular technique/ protocol but it does not hold water when the claim of a *"new paradigm"* in vitrification was put on the table seven years after the Isachenkos' paper, with essentially the same claim that had been published [Isachenko *et al.*, 2004b]. That new paradigm was indeed established but

Note that the role of ultrafast warming during kinetic vitrification had been known at some extend before so neither of the authors (the Isachenkos or Mazur & Seiko) can claim the absolute priority. In case with the crucial role of endogenous proteins and other high molecular weight components for the intracellular *kinetic* vitrification of spermatozoa, Katkov and colleagues clearly presented this idea (and indirect proof of it) in 2003. Therefore, any attempts to completely ignore that fact by Morris and colleagues and to position themselves as *"pioneers"* of this idea much later can be considered as blunt

scientists reported in 2010 on Cryo-2010 in Bristol, UK and other publications.

**John Morris:**

(**CRYO-2010**).

*Cryobiology*, **64**:71-80).

*- "It is generally assumed* that the intracellular environment of sperm has a low water content coupled with high protein levels. These data demonstrates that it heterogeneous nucleation sites are absent in that intracellular vitrification can occur:"

**- "We demonstrate t**hat the high intracellular protein content together with the osmotic shrinkage associated with extracellular ice formation leads to intracellular vitrification of spermatozoa during cooling" (Morris et al, 2011,


*WARMING rate is much more critical in "diluted" vitrification solutions than the cooling rate* (**CRYO-2010)**, [Mazur & Seki, 2011]

**Peter Mazur and Shinsuke Seki:** 

**V. & E. Isachenkos and I.I. Katkov:** Factors that may enhance intracellular

**V. & E. Isachenkos** and **I.I. Katkov:** 

it was done in 2002-4, not in 2010-11!

… *As a result, we can speculate that we were able to achieve intracellular vitrification of the human spermatozoa even at such a low range cooling rate. ... However, as we discuss below, our method of instant thawing seemed to prevent cell damage even after relative slow freezing in liquid nitrogen vapor.* [Isachenko

Crucial role of fast warming

*et al.*, 2004b]

plagiarism.

…*Cells naturally contain high concentrations of proteins, which help in vitrifcation… this would enhace both the viscosity and Tg of the intracellular cytosol of permatozoa .* [Isachenko et al., 2003].

vitrification of human sperm:

Fig. 6. Different techniques for kinetic vitrifcation of human sperm developed by the Isachenkos and colleagues: A: copper or nylon cryo loop (pellicle); B: a modification of a droplet technique; C; open-pulled straw; D; straw-in- straw. See [Isachenko *et al., 2*007] for details.

The method and the scientist themselves were first dismissed, than ignored, than … ignored again. **Table 1** represents just two examples when the Isachenkos published their paper, and other cryobiologists, who came to the same conclusions, namely:


Thus, both cryobiologists have reported similar findings as the Isachenkos observations, but they unfortunately fell short of mentioning Isachenkos in their own publications and presentations (e.g., in Cryo-2010 in Bristol), which might have made looking their observations (that were solid, of course) for an unfamiliar reader as "pioneering" or even as *"a new paradigm for cryopreservation by vitrifcation"* [Mazur & Seki, 2011]. The argument *"that paper by the Isachenkos et al. was not citable because the effect of the warming rates was not* 

A B

C D

other cryobiologists, who came to the same conclusions, namely:

sperm is also questioned in [Mazur & Koshimoto, 2002].

very fast warming is essential for kinetic VF [Mazur & Seki, 2011]

details.

Fig. 6. Different techniques for kinetic vitrifcation of human sperm developed by the Isachenkos and colleagues: A: copper or nylon cryo loop (pellicle); B: a modification of a droplet technique; C; open-pulled straw; D; straw-in- straw. See [Isachenko *et al., 2*007] for

The method and the scientist themselves were first dismissed, than ignored, than … ignored again. **Table 1** represents just two examples when the Isachenkos published their paper, and



Thus, both cryobiologists have reported similar findings as the Isachenkos observations, but they unfortunately fell short of mentioning Isachenkos in their own publications and presentations (e.g., in Cryo-2010 in Bristol), which might have made looking their observations (that were solid, of course) for an unfamiliar reader as "pioneering" or even as *"a new paradigm for cryopreservation by vitrifcation"* [Mazur & Seki, 2011]. The argument *"that paper by the Isachenkos et al. was not citable because the effect of the warming rates was not* 


Table 1. Comparison of statements published by Isachenkos et al in 2003-4 and by other scientists reported in 2010 on Cryo-2010 in Bristol, UK and other publications.

*thoroughly investigated"* makes sense in the matter of describing a particular technique/ protocol but it does not hold water when the claim of a *"new paradigm"* in vitrification was put on the table seven years after the Isachenkos' paper, with essentially the same claim that had been published [Isachenko *et al.*, 2004b]. That new paradigm was indeed established but it was done in 2002-4, not in 2010-11!

Note that the role of ultrafast warming during kinetic vitrification had been known at some extend before so neither of the authors (the Isachenkos or Mazur & Seiko) can claim the absolute priority. In case with the crucial role of endogenous proteins and other high molecular weight components for the intracellular *kinetic* vitrification of spermatozoa, Katkov and colleagues clearly presented this idea (and indirect proof of it) in 2003. Therefore, any attempts to completely ignore that fact by Morris and colleagues and to position themselves as *"pioneers"* of this idea much later can be considered as blunt plagiarism.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 27

**0%**

**0%**

**20%**

**40%**

**Acrosomal Integrity**

**60%**

**80%**

**100%**

**20%**

**40%**

**60%**

**Motility**

**80%**

**100%**

**p<0.03**

**Fresh Vitrification (Isachenko) Slow Freeze**

**Human Mouse Rat**

**Fresh Vitrification (Isachenko) Slow Freeze**

**Human Mouse Rat**

Fig. 7. Progressive motility (**left**) and acrosomal integrity (**right**) of 3 species of sperm frozen by conventional slow-freezing protocols in the media customized for different species (**green**) and by an identical protocol of vitrification [Isachenko et al., 2008] by

(**red**) directly into LN2. Two methods of cryopreservation are compared.

quenching droplets in PBS containing 20% human serum albumin (HSA) and 0.25 M sucrose

**p<0.01**

**p<0.04**

#### **7.2 Kinetic vitrification of sperm of** *other* **vertebrates: history of success and stories of failure**

As we have mentioned, kinetic VF of human sperm in all its varieties shown on Fig. 6 seemed to be working equally well; however, when we tried the "droplet method" described in [Isachenko *et al.,* 2008] (20 μL droplets of swam-up washed sperm supplemented by 0.25 M sucrose) on model animals (rodent spermatozoa, the results (Figs. 7) were not so pronounced. So, while it worked well for human sperm, the droplet kinetic VF did not work so well for mouse sperm, and it worked poorly (at a much lower survival rate than conventional slow freezing) on rat sperm. Note that both rats and mice sperm have larger and apparently more watery heads.

But still, the Isachenkos' method worked in general so Celltronix and Kharkov Zoo launched 2 field expeditions (with the participation of a Moscow Zoo's specialist) for freezing polar bear (Ursus maritimus) sperm (in a distant Russian zoo) and sperm of gyrfalcons (Falco rusticolus), golden eagles (*Aquila chrysaetos*) and Eastern imperial eagles (*Aquila heliaca*) in the Russian Raptor Breeding Center Galichya Gora near Voronezh.

For the polar bear, for which sperm, to our knowledge, had not been frozen yet, the basic slow protocol developed for spectacled bear (*Tremarctos ornatus*) [Erokhin *et al.,* 2007] was used. That protocol worked quite poorly, vitrification protocol was even worse.

For the all 3 raptor species, gyrfalcon, the golden and the imperial eagles, slow freezing (using the slow freezing protocol in [Blanco et al., 2000]) worked very poorly in our hands despite the fact that artificial insemination with fresh sperm is a routine and successful procedure in that Center. And finally, kinetic vitrification using the Isachenko's "droplet" method failed completely.

After several sleepless nights of thinking what went wrong besides our insufficient experience with freezing the raptor sperm (my counterpart had frozen crane sperm before), difficulties related to small volume and a lot of fecal particles and urine in sperm, I.I. Katkov realized that the sperm of those species was fundamentally different in geometry from that of human sperm: their heads were much larger, and they looked much more watery, less condensed than the compact human portions. The rodent sperm heads were also relatively large, but those species, where we failed, the heads apparently contained much more water and presumably less so called "inactive osmotic volume", which means the concentration of internal proteins, sugars, nucleotides and other "internal endogenous vitrificants" was much lower in the polar bear spermatozoa, and especially in the sperm of the raptors. And according to the thermodynamic of the glassy state, as lower concentration of vitrificants the faster you need to cool the cells. From the personal communication with the Isachenkos several years before, it was known that kinetic vitrification of oocytes and embryos without cryoprotectants had been failed completely even with the smallest drops. And those cells have the ratio internal vitrificants: water about 7-9 lower than in human sperm as its osmotically active volume (i.e. water per se) is about 75%, while in human sperm it is only 25%. That meant that we just did not have sufficient cooling speed to vitrify those species!! That crystallized the hypothesis that if we would cool it fast enough, faster than the critical rates of cooling and warming for the most watery cells, we can vitrify all cells with the same protocol. That is how the concept of the Universal Cryopreservation Protocol was born (published first in the "Embryomail" in the spring of 2010).

**7.2 Kinetic vitrification of sperm of** *other* **vertebrates: history of success and stories** 

As we have mentioned, kinetic VF of human sperm in all its varieties shown on Fig. 6 seemed to be working equally well; however, when we tried the "droplet method" described in [Isachenko *et al.,* 2008] (20 μL droplets of swam-up washed sperm supplemented by 0.25 M sucrose) on model animals (rodent spermatozoa, the results (Figs. 7) were not so pronounced. So, while it worked well for human sperm, the droplet kinetic VF did not work so well for mouse sperm, and it worked poorly (at a much lower survival rate than conventional slow freezing) on rat sperm. Note that both rats and mice sperm have

But still, the Isachenkos' method worked in general so Celltronix and Kharkov Zoo launched 2 field expeditions (with the participation of a Moscow Zoo's specialist) for freezing polar bear (Ursus maritimus) sperm (in a distant Russian zoo) and sperm of gyrfalcons (Falco rusticolus), golden eagles (*Aquila chrysaetos*) and Eastern imperial eagles

For the polar bear, for which sperm, to our knowledge, had not been frozen yet, the basic slow protocol developed for spectacled bear (*Tremarctos ornatus*) [Erokhin *et al.,* 2007] was

For the all 3 raptor species, gyrfalcon, the golden and the imperial eagles, slow freezing (using the slow freezing protocol in [Blanco et al., 2000]) worked very poorly in our hands despite the fact that artificial insemination with fresh sperm is a routine and successful procedure in that Center. And finally, kinetic vitrification using the Isachenko's "droplet"

After several sleepless nights of thinking what went wrong besides our insufficient experience with freezing the raptor sperm (my counterpart had frozen crane sperm before), difficulties related to small volume and a lot of fecal particles and urine in sperm, I.I. Katkov realized that the sperm of those species was fundamentally different in geometry from that of human sperm: their heads were much larger, and they looked much more watery, less condensed than the compact human portions. The rodent sperm heads were also relatively large, but those species, where we failed, the heads apparently contained much more water and presumably less so called "inactive osmotic volume", which means the concentration of internal proteins, sugars, nucleotides and other "internal endogenous vitrificants" was much lower in the polar bear spermatozoa, and especially in the sperm of the raptors. And according to the thermodynamic of the glassy state, as lower concentration of vitrificants the faster you need to cool the cells. From the personal communication with the Isachenkos several years before, it was known that kinetic vitrification of oocytes and embryos without cryoprotectants had been failed completely even with the smallest drops. And those cells have the ratio internal vitrificants: water about 7-9 lower than in human sperm as its osmotically active volume (i.e. water per se) is about 75%, while in human sperm it is only 25%. That meant that we just did not have sufficient cooling speed to vitrify those species!! That crystallized the hypothesis that if we would cool it fast enough, faster than the critical rates of cooling and warming for the most watery cells, we can vitrify all cells with the same protocol. That is how the concept of the Universal Cryopreservation Protocol was born

(*Aquila heliaca*) in the Russian Raptor Breeding Center Galichya Gora near Voronezh.

used. That protocol worked quite poorly, vitrification protocol was even worse.

(published first in the "Embryomail" in the spring of 2010).

**of failure** 

larger and apparently more watery heads.

method failed completely.

Fig. 7. Progressive motility (**left**) and acrosomal integrity (**right**) of 3 species of sperm frozen by conventional slow-freezing protocols in the media customized for different species (**green**) and by an identical protocol of vitrification [Isachenko et al., 2008] by quenching droplets in PBS containing 20% human serum albumin (HSA) and 0.25 M sucrose (**red**) directly into LN2. Two methods of cryopreservation are compared.

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 29

Fig. 8C. An attempt to vitrify sperm of the golden eagle, **from left to right**: I.I.K. with the bird; sperm retrieval process; fresh; slow frozen; and vitrified sperm. Slow frozen sperm

**8***.* **Conclusion:** *"Race for the Pace"***: Is the universal cryo-protocol possible?**  The universal cryoprotocol, that would fit *all* types of cells, at least if they are in suspension on make a thin layer, would be the *Holy Grail* of cryobiology. Here is our hypothesis for

1. Every cell has its own critical rates of cooling and thawing, at which and higher the cell can be vitrified during cooling (*Bcr\_cool*) and will not devitrify during warming (*Bcr\_warm*) *without* any external "cryoprotectants" (they must be called "vitrificants" in this case). Or it might be just that non-lethal ice (i.e. cubical vs. hexagonal "killer ice") is formed during cooling and its transformation (recrystallization) to hexagonal type is precluded during warming. In any case, at rates higher than those two *Bcr*'s, the cell will survive

2. Those rates are substantially *lower* than predicted by the contemporary theories (Fahy and Rall, Boutron's work, Cravalho 's school: Toner, Karlsson, *et al*). We will not go into the details of the thermodynamics of the glassy state but the three main reasons are: i) presence of the internal cell vitrificants with high *Tg*; ii) small compartmentalized intracellular milieu; iii) no "true" extracellular VF is needed for survival as the cell has no time and shrink at such fast time. In any scenario, the cell *survives* if the pace of

cooling and warming is higher than those two *Bcr***'**s, and that is what matters. 3. The distribution and average values of those *Bcr***'**s 's depend on the species of cells, particularly on the abundance of endogenous vitrificants, how "watery" those cells are, the level of compartmentalization, the size of the compartments, etc. It may well be that the same species (such embryos) might have *very* different *Bcr***'s** at different stages of

protocol worked poorly, *kinetic VF failed*.

without any exogenous compounds.

consideration [Katkov, 2010]:

their development.

Fig. 8A. An attempt to vitrify sperm of the polar bear, **from left to right**: sperm retrieval; fresh; slow frozen; and vitrified sperm. Slow frozen sperm protocol worked poorly, *kinetic VF failed*.

Fig. 8B. An attempt to vitrify sperm of the gyrfalcon, **from left to right**: I.I.K. with the bird; sperm retrieval process; fresh; slow frozen; and vitrified sperm. Slow frozen sperm protocol worked poorly, *kinetic VF failed*.

Fig. 8A. An attempt to vitrify sperm of the polar bear, **from left to right**: sperm retrieval; fresh; slow frozen; and vitrified sperm. Slow frozen sperm protocol worked poorly, *kinetic* 

Fig. 8B. An attempt to vitrify sperm of the gyrfalcon, **from left to right**: I.I.K. with the bird; sperm retrieval process; fresh; slow frozen; and vitrified sperm. Slow frozen sperm protocol

*VF failed*.

worked poorly, *kinetic VF failed*.

Fig. 8C. An attempt to vitrify sperm of the golden eagle, **from left to right**: I.I.K. with the bird; sperm retrieval process; fresh; slow frozen; and vitrified sperm. Slow frozen sperm protocol worked poorly, *kinetic VF failed*.
