**1. Introduction**

This as well as two other related Chapters, by Isachenko *et al.* and Moskovtsev *et al*., open this Book neither accidentally nor by the Editor's preferences to his friends and collaborators; the reasons, in fact, lie quite deeper:

Why *sperm?* Cryobiology had actually started from freezing sperm. We will skip all those very early anecdotes but should mention the Spallanzani attempt to freeze frog semen in the 18th century [Spallanzani, 1780]. Cryobiology as a science started with revolutionizing work of Father Luyet and other scientists of the late 1930's and 1940's, who we can collectively call *"the pioneers of the cryobiological frontiers"* (see the following sub-Chapter). There were several reasons why sperm was chosen, which included easiness in obtaining the samples, clear evidence of viability (moving – not moving, though later it was figured that everything was not so easy in this sophisticated living *"cruise missile"*), and importance for the farming industry with the emergence of systematic selective breeding (especially in cattle) with a powerful tool – artificial insemination (AI). AI started with the revolutionary work of W. Heape, I.I. Ivanov and other scientists at the dawn of the 20th century and was further developed by V.K. Milovanov in the 1930's as a viable breeding technology (see [Foote,

 \* V.F. Bolyukh2, O.A. Chernetsov3, P.I. Dudin4, A.Y. Grigoriev5, V. Isachenko6, E. Isachenko6,

A.G.-M. Lulat7, S.I. Moskovtsev7,8, M.P. Petrushko9, V.I. Pinyaev9, K.M. Sokol10, Y.I. Sokol2, A.B. Sushko3 and I. Yakhnenko1

*<sup>1</sup> CELLTRONIX and Sanford-Burnham Institute for Medical Research, San Diego, California, USA 2 Kharkov National Technical University "KhPI", Kharkov, Ukraine 3 Animal Reproduction Center, Kulinichi, Kharkov region, Ukraine* 

*<sup>4</sup> Raptor Restoration and Reintroduction Program, National Reserve "Galichya Gora", Voronezh region, Russia 5 Kharkov Zoo, Kharkov, Ukraine* 

*<sup>6</sup> Dept. Obstetrics and Gynecology, Ulm University, Germany 7 CReATe Fertility Center, Toronto, Ontario, Canada* 

*<sup>8</sup>Dep. Obstetrics and Gynecology, Toronto University, Toronto, Ontario, Canada 9 ART Clinic, Kharkov, Ukraine* 

*<sup>10</sup> Kharkov National Medical University, Kharkov, Ukraine* 

*<sup>\*\*</sup>* Corresponding Author

Kinetic Vitrification of Spermatozoa of Vertebrates: What Can We Learn from Nature? 5


**In this Chapter**, we summarize the basic thermodynamical and biophysical distinctions between K-VF *,* E-VF , slow freezing (SF), analyze present and predict future developments that will widen the K-VF niche, and hypothesize why K-VF of some species of sperm was more successful than the others. We then briefly explore our idea that with the development of a new generation of hyper-fast cooling devices (up to several hundred of thousand OC/min), we will witness the "*Race for the Pace*" for the *Universal Cryopreservation Protocol* 

**2. Brief history of kinetic vitrification of sperm and cryobiology in general** 

In the dawn of cryopreservation, vitrification of small samples by ultra-fast cooling (tens of thousands OC/min) without additional thickening and ice-blocking agents (VFAs), which is

without any exogenous VFA's that can be applicable to *any* cell type.

**2.1 Early attempts of** *kinetic* **vitrification of sperm and other cells** 

publications.

Chapters at the spearhead of the Book.

**related to the goal of this Chapter** 

incorrect terminology, and as the result, to be a target of criticism in following

2002] and [Milovanov, 1962] for detailed history of AI). Whatever case(s) for such specific interest to freezing sperm had been, it was the first subject of systematic research in cryobiology. For a long time after the 1940's, cryopreservation (CP) of sperm would be overshadowed by successes in CP of other types of cells: peripheral blood, blood, embryos, cord blood, stem cells, and other cells, tissues and organs. However, the recent progress and rejuvenation of the old method of sperm vitrification (see following Chapters by Isachenko and Moskovtsev) makes us to believe that it can bring a new shift in the cryobiological paradigm, which we will discuss later in this Chapter.

Why *vitrification?* As we will discuss below, the only method of stable and long-term (practically infinite) preservation and storage of any perishable biological materials, particularly cells, (a.k.a. "*biostabilization"*) is to keep them in the glassy (vitreous) state. This was clearly understood by Father Luyet when he titled his pioneering work "The *vitrification* of organic colloids and of protoplasm" and "Revival of frog's spermatozoa *vitrified* in liquid air" [Luyet & Hodapp, 1938; Luyet, 1937]. He and other *"pioneers of the cryobiological frontiers"*  clearly understood 70 years ago that only glassy state would insure stable and non-lethal preservation of cells. With time, we saw the development of a variety of biopreservation methods,such as slow freezing (which, as we will see below, is just a way of achieving glassy state inside the cells and within their close vicinity - cells cannot live neither within ice without a glassy border between cells and ice, or with ice within them). Another method is *equilibrium* vitrification with large amounts of exogenous thickeners (vitrification agents, or VFAs). Eventually, many cryobiologists, especially the new generation and many practitioners, have forgotten that all those methods are basically different ways of achieving vitrification of the intracellular milieu (or at least, without the formation of intracellular types of ice that kill the cells) and the cell's close extracellular vicinity. This has led to several common misconceptions:


2002] and [Milovanov, 1962] for detailed history of AI). Whatever case(s) for such specific interest to freezing sperm had been, it was the first subject of systematic research in cryobiology. For a long time after the 1940's, cryopreservation (CP) of sperm would be overshadowed by successes in CP of other types of cells: peripheral blood, blood, embryos, cord blood, stem cells, and other cells, tissues and organs. However, the recent progress and rejuvenation of the old method of sperm vitrification (see following Chapters by Isachenko and Moskovtsev) makes us to believe that it can bring a new shift in the cryobiological

Why *vitrification?* As we will discuss below, the only method of stable and long-term (practically infinite) preservation and storage of any perishable biological materials, particularly cells, (a.k.a. "*biostabilization"*) is to keep them in the glassy (vitreous) state. This was clearly understood by Father Luyet when he titled his pioneering work "The *vitrification* of organic colloids and of protoplasm" and "Revival of frog's spermatozoa *vitrified* in liquid air" [Luyet & Hodapp, 1938; Luyet, 1937]. He and other *"pioneers of the cryobiological frontiers"*  clearly understood 70 years ago that only glassy state would insure stable and non-lethal preservation of cells. With time, we saw the development of a variety of biopreservation methods,such as slow freezing (which, as we will see below, is just a way of achieving glassy state inside the cells and within their close vicinity - cells cannot live neither within ice without a glassy border between cells and ice, or with ice within them). Another method is *equilibrium* vitrification with large amounts of exogenous thickeners (vitrification agents, or VFAs). Eventually, many cryobiologists, especially the new generation and many practitioners, have forgotten that all those methods are basically different ways of achieving vitrification of the intracellular milieu (or at least, without the formation of intracellular types of ice that kill the cells) and the cell's close extracellular vicinity. This has led to several common misconceptions: - The fact that permeable substances such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG or PrOH) and some other small permeable compounds play absolutely different roles during *slow freezing*, when they serve mainly as osmotic buffers and during *vitrification* (VF), when they play the role of thickeners so they increase viscosity and deplete growth and propagation of ice. As a result, in both cases, these substances are called "cryoprotective agents" (CPA's) across the board even though the concentrations used, the modes of addition and elution, and the mechanisms of action are very different for the cases of *slow freezing* (SF) *vs*. *equilibrium* vitrification (E-VF) and *kinetic* vitrification (K-VF) (we will explain the difference between E-VF and K-VF later). We prefer to distinguish these two roles and call 10% of DMSO used for *slow freezing* of stem cells as *"CPA"* and 40% of DMSO used for *equilibrium* VF of embryos of kidneys as *"VFA"*. As we can see however, for *kinetic* VF, even 10% of glycerol can help vitrify the cells and can be used as the vitrification agent (with some


paradigm, which we will discuss later in this Chapter.

reservation).

incorrect terminology, and as the result, to be a target of criticism in following publications.


Why *kinetic?* As we will also discuss below, the modern shift from Fahy's *equilibrium* back to Luyet's *kinetic* vitrification has brought not only clear technical advantages and better survival of oocytes and embryos. The resurrection and successful re-emergence by the Isachenkos of the Luyet's method in regards to the very subject he and other *"pioneers of the cryobiological frontiers"* attempted to preserve more than 70 years ago - the sperm, has not only brought a simple and convenient technique to the field of assisted reproduction (human spermatozoa first, then animal ones followed). As we can see later in this Chapter, both success of K-VF for some species of sperm and failure of the same method for the others would prompt us to a more general idea: the *"Universal Cryopreservation Protocol"*, which could have a much broader impact and if realized physically by a new type of cryogenic devices that would insure hyperfast cooling and warming, it would shift the whole cryopreservation paradigm. We feel that we will soon witness some sort of a *"Kinetic Vitrification Spring"* as to draw a political analogy, and that is why we have put these three Chapters at the spearhead of the Book.

**In this Chapter**, we summarize the basic thermodynamical and biophysical distinctions between K-VF *,* E-VF , slow freezing (SF), analyze present and predict future developments that will widen the K-VF niche, and hypothesize why K-VF of some species of sperm was more successful than the others. We then briefly explore our idea that with the development of a new generation of hyper-fast cooling devices (up to several hundred of thousand OC/min), we will witness the "*Race for the Pace*" for the *Universal Cryopreservation Protocol*  without any exogenous VFA's that can be applicable to *any* cell type.
