Cryobiological determinants of frozen semen quality, with special reference to stallion

Animal Reproduction Science 107 (2008) 276–292

Cryobiological determinants of frozen semen quality, with special reference to stallion夽 H. Sieme a,∗ , R.A.P. Harrison b , A.M. Petrunkina a,c a

Clinic for Horses, Reproductive Unit of Clinics, University of Veterinary Medicine, Hannover, Germany b 11 London Road, Great Shelford, Cambridge, UK c Cambridge Institute for Medical Research, University of Cambridge, UK Available online 9 May 2008

Abstract Success in cryopreserving stallion semen has been very variable. Several different freezing regimes have been published. However, because extenders and procedures used in each regime have differed, direct comparison of these techniques has been very difficult, and controlled studies comparing different techniques have not been reported. A number of different factors affect sperm cryosurvival. In this article we review briefly current cryopreservation procedures for stallion semen, and then in more detail cryobiological determinants of sperm function, and mechanisms of cryoinjury and cryoprotectant action. Specific attention is given to data relating to stallion sperm. The complexity of sperm cell biology is believed to be an important factor when developing improvements in stallion semen cryopreservation. It may be assumed that impairment of cell function resulting from cold and osmotic shock is a main source of stallion sperm sensitivity to conventional freezing procedures. Further physiological studies on stallion sperm are required to understand the mechanisms by which cryopreservation alters sperm function and influences selection of sperm with higher fertilizing potential. Such studies should focus especially on the processes involved in sperm volume regulation, sperm–oviduct interaction, capacitation and cellular signalling, and on the alterations in these processes caused by cryopreservation. © 2008 Elsevier B.V. All rights reserved. Keywords: Stallion semen; Cryopreservation; Osmotic properties; Cryoinjury; Sperm function; Male heterogeneity

夽 This paper is part of the Special issue entitled “Proceedings of the 5th International Symposium on Stallion Reproduction”, Guest Edited by Terttu Katila. ∗ Corresponding author at: Clinic for Horses, Reproductive Unit of Clinics, University of Veterinary Medicine Hannover, B¨unteweg 15, D-30559 Hannover, Germany. Tel.: +49 511 953 8530; fax: +49 511 953 82 8530. E-mail address: [email protected] (H. Sieme).

0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2008.05.001

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1. Current techniques of cryopreservation of stallion semen It is generally believed that the key to cryopreservation success of stallion semen is the individual stallion itself. Stallions have shown a particularly high degree of individual variation with respect to the cryosurvival of their sperm. It has been estimated that ∼20% of stallions produce semen that freeze well, 60% freeze acceptably and 20% freeze poorly (Vidament et al., 1997). Stallions that are satisfactorily fertile under normal field conditions can produce semen that after freezing and thawing produces very low pregnancy rates. The mechanisms underlying the differences in cryosensitivity between different individuals have yet to be elucidated. Such differences could be genetic in origin, and genetic selection of stallions for successful freezing could be a possibility. On the other hand, the difference might be non-genetic and in this regard it would be particularly desirable to be able to apply assays of sperm function before and after freezing which correlate well with either semen freezability or stallion fertility. Unfortunately, such assays are not currently available (Katila, 2001; Kuisma et al., 2006). Comparing protocols from different countries, methods for freezing stallion semen and instructions for insemination of mares with frozen–thawed semen are far from standardized (for review: Samper and Morris, 1998). The lack of standardization is demonstrated by the several differences in processing stallion semen for cryopreservation using different methods for packaging and thawing (Table 1). With regard to post-thaw sperm quality cryopreserved stallion semen is best prepared during the non-breeding season (Janett et al., 2003). Stud farms should take into consideration many practical matters (e.g. sexual rest, individual characteristics of stallions, and hygienic conditions) that precede the freezing campaign. After long sexual rest, extragonadal sperm reserves should be depleted by repeated collections until good sperm quality is established. For preparing frozen semen, it would be preferable to make the semen collection interval at least 48 h, though this should be adapted to the individual stallion (Sieme et al., 2004). Semen collection should be carried out using a suitable technique (type of artificial vagina, lubricant, collector) and a fixed time interval between collections to minimize intra-individual differences between ejaculates. Current freezing protocols for stallion semen involve a two-step dilution procedure in which semen is first diluted with a primary extender, centrifuged and then diluted a second time prior to freezing in an extender containing a cryoprotectant. The first dilution employs either saline/sugar extenders or skim milk extenders used to dilute fresh semen. The dilution rate is either 1:1 or the semen is diluted to a concentration of ∼50 million spermatozoa/ml. The success of centrifugation depends on duration (10–15 min) and centrifugation force (350–700 × g). To increase sperm recovery, the use of high-speed centrifugation (20 min, 1000 × g) through a liquid cushion has been introduced into laboratory practice (Knop et al., 2005); this technique had no detrimental effect on fertility (Ecot et al., 2005). Moore et al. (2005) demonstrated the deleterious effect of seminal plasma on stallion spermatozoa during cryopreservation. However, retention of 5–20% seminal plasma in the suspension after centrifugation has been considered to be essential for cryosurvival. Glycerol has been used almost universally as the cryoprotectant, although it has been recently reported that other cryoprotectants such as dimethyl sulfoxide, ethylene glycol, methyl formamide or dimethyl formamide may yield similar or superior results (Squires et al., 2004; Alvarenga et al., 2005). Fertility of stallion semen frozen with either glycerol or dimethyl formamide was not different (Vidament et al., 2002). The composition of the freezing extender may influence the length of the cooling phase required before freezing. Essentially the semen should cool slowly from room temperature to 5 ◦ C

278

Source

Dilution

Centrifugation extender

Tischner (1979)

Collection of sperm-rich fraction

He (1986) Martin et al. (1979)

1:1 1:1

11% sucrose Glucose–EDTA

Loomis et al. (1983) Cochran et al. (1984)

1:1 50 × 106 sp./ml

Palmer (1984)

1:4

H˚aa˚ rd and H˚aa˚ rd (1991)

50–80 × 106 sp./ml

Vidament (2005)

2.5 × 109 sp./50 ml tube

Freezing extender

Glycerol concentration (%)

Packaging

Freezing

Thawing

Lactose–EDTA–EY

3.5

7–9 min, N2 vapour

40 ◦ C, 50 s

350–450 g, 12 min 1000 g, 10 min

Sucrose–milk–EY Merck–lactose–EY

4–5 5

N2 vapour 20 min, N2 vapour

Sucrose–milk 50 ◦ C, 40 s

Glucose–EDTA Citrate–EDTA (20 ◦ C), cushion

650 g, 15 min 400 g, 15 min

EDTA–lactose–EY Lactose–EDTA–EY

5 5

Aluminium-tubes (20–25 ml) Glass-vials (1 ml) Macrot¨ub® (4.0 ml straw) 0.5 ml straw

38 ◦ C, 30 s 37 ◦ C, 30 s or 75 ◦ C, 7 s

INRA-82-EY → +4 ◦ C/1 h Citrate–EDTA (30 ◦ C), cushion

600 g, 10 min

INRA-82–EY

2.5

400 g, 15 min

Lactose–EDTA

5

600 g, 10 min

INRA-82–EY

2.5

10 min, N2 vapour +20 → −15 ◦ C: 10 ◦ C/min; −15 → −120 ◦ C: 25 ◦ C/min +4 → −140 ◦ C: 60 ◦ C/min +20 → −10 ◦ C: 10 ◦ C/min; −10 → −140 ◦ C: 25 ◦ C/min →+4 ◦ C: 1 h 15 min; +4 → −140 ◦ C: 40–60 ◦ C/min

UHT skim milk (37 ◦ C)

Centrifugation

37 ◦ C, 30 s 75 ◦ C, 7 s then 35 ◦ C, 10–30 s

37 ◦ C, 30 s

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Table 1 Processing methods used for cryopreservation of stallion semen

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(0.05 ◦ C/min) before storage at 5 ◦ C under anaerobic conditions. In most laboratories, the cooled semen is put into 0.5 straws at 5 ◦ C (Vidament et al., 2000). Depending on the method of processing and storage, the optimal freezing rate for stallion semen has been reported to range between 20 and 100 ◦ C/min (Cristanelli et al., 1985; Devireddy et al., 2002a,b). Alternative techniques to freeze stallion semen have been published recently, e.g. directional freezing. In directional freezing, after an initial seeding stage, the semen sample is advanced at a constant velocity through a linear temperature gradient. In this way, the ice crystal propagation can be controlled so as to optimize crystal morphology, achieve continual seeding and a homogenous cooling rate throughout the entire freezing process (Zirkler et al., 2005; Saragusty et al., 2007). Although vitrification of human sperm has been described (Isachenko et al., 2004), the authors are not aware that a vitrification procedure has been published as an alternative to conventional cryopreservation of stallion semen. 2. Cryobiological determinants of sperm function Cryopreservation methodology for spermatozoa has hitherto been developed largely by empirical means. However, many fundamental biophysical studies have sought to understand the general cellular processes involved in freezing and thawing. From these studies, methods to model cryobiological processes mathematically have been developed, in order to deduce optimal freezing and thawing rates and thereby improve cryopreservation methodologies. In the first part of this section, the qualitative aspects of these processes will be outlined. The second part will describe and comment on the quantitative parameters used to perform mathematical modelling. The section will focus on spermatozoa from large animals, especially from stallion. For further details regarding mathematical modelling and the relevant equations, the reader should consult earlier reviews (e.g. Pegg, 2002; Fuller and Paynter, 2004; Fuller et al., 2004; Petrunkina, 2007). 2.1. Mechanisms of cryoinjury and cryoprotectant action The quality of frozen–thawed sperm will depend greatly on the cells’ ability to withstand cryopreservation processes without loss of major cellular functions (such as viability, motility and more complex physiological functions). In a biophysical approach to cryobiology, the behaviour of cells is predicted based on models of mass solute and water transfer at various temperatures. The process of cryopreservation leads to morphological and functional damage of cells largely because it is associated with considerable osmotic stresses. The osmotic shock itself can be caused by a variety of factors other than the nominal osmolalities of the media. Osmotic injury occurs when cryopreserved spermatozoa are diluted into iso-osmotic media, or when spermatozoa are placed in the female reproductive tract. The injury stems from the hyperosmolarity of extenders, the changes in relative solute concentration during freezing/thawing, and the differences in the relative permeabilities of water and impermeant and permeant solutes. During freezing, cells became dehydrated and shrink; during thawing, as water re-equilibration takes place, they are submitted to hypotonic challenge, a process which is associated with a volume expansion. The shrinkage and swelling of cells are limited by their capacity to withstand such changes. Damage will occur beyond certain levels of shrinkage or swelling (the overall range between these levels is known as the maximal cell volume excursion) as discussed below. The optimal equilibrium freezing rate found for a given cell

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type must result in a tolerable volume excursion, i.e. a range of swelling and shrinkage which does not lead to the loss of membrane integrity and impaired cell function (e.g. motility for spermatozoa). Both too slow and too rapid freezing were found to be associated with lethal cryoinjury (Mazur et al., 1972). Therefore, it is important to understand and to investigate the kinetics of cellular water loss/volume excursions, using the quantitative parameters discussed below. Rapid freezing will lead to intracellular ice formation because higher cooling rates lead to supercooling of cells, either above or below their nucleation temperature. Low hydraulic permeability represents another critical factor—water will not be able to leave the cells in sufficient quantity at higher freezing rates. In this case water will be trapped in the cell, and damaging ice crystals will be formed within (Muldrew and McGann, 1990, 1994). However, if freezing progresses at very slow rates, the dehydration will take place over a longer time period. During this slow freezing, the osmolality of the external medium will increase so that the cells will be exposed to high salt concentrations in the external medium, whence they will dehydrate completely to a degree of shrinking associated with fatal cellular disruption (Mazur et al., 1972). Inability of cells to recover their volume after thawing will result in dysfunction and cell death. Cryoinjury due to the sensitivity of cells to high solute concentrations is referred to as a ‘solution effect’. The freezing point of a solution is primarily dependent on the concentration of solute(s) which it contains. The cellular state and water balance in cells during freezing and thawing is determined by the colligative properties of the aqueous solution, i.e. by the properties which depend on concentrations of solutes rather than on behaviour of individual chemical compounds. Cryoprotective agents that diminish cell damage are usually divided into two general classes related to their ability to move across the cell membrane: permeant and impermeant. Their mechanisms of action are different. Cryoprotection by permeant agents can be explained by their colligative properties: they lower the temperature at which cells are exposed to a critical salt concentration (Lovelock, 1953; McGann, 1978). Impermeable agents, on the other hand, are thought to dehydrate cells, allowing them to be cooled rapidly before lethal injury due to solution effects takes place (McGann, 1978). Freezing rates should therefore be slow enough to allow the cells to minimize chemical potential and osmolality gradients across the plasma membrane and to dehydrate without being exposed to critical hypertonicity. This ‘equilibrium’ freezing leads to tolerable cell shrinkage, to an increase in the intracellular chemical potential, and to depression of the freezing temperature. If water transport proceeds at a suitable rate, the amount of intracellular water will decrease, and if a permeant cryoprotective agent is used, the freezing point will be depressed. In doing that, the potential risk of intracellular ice formation will be reduced, so that only extracellular ice will be formed. These considerations explain why the characteristic shape of the curve plotting cell survival against freezing rate is an inverted ‘U’, with typically a critical cooling rate for maximum survival (Mazur et al., 1972). However, the effects of cryoprotectants are more complex than can be explained by thermodynamics and solution theory. In high concentrations, they can produce lethal osmotic effects during their addition and removal (hyperosmolality) and they can be toxic (Woods et al., 2004). Initial cellular shrinking (due to dehydration) caused by molar concentrations of cryoprotectant, and subsequent swelling during its removal, can both result in damage if the maximal volume excursion is exceeded. Whether the effects of a given cryoprotective agent are negative or positive can be greatly dependent on the kinetics of exposure.

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2.2. Biophysical parameters relevant to cryopreservation and their use in mathematical modelling Mathematical modelling is used to try to predict optimal cooling and thawing rates. The models involve the following parameters: • • • • • • • •

cell size (cell volume V and surface area to volume ratio A/V); osmotic behaviour and tolerable excursions; fractional osmotically inactive cell volume Vb ; water permeability Lp (hydraulic conductivity) of the plasma membrane; permeability coefficients (Ps ) of relevant solutes; reflection coefficient σ of these solutes; activation energy Ea ; initial concentration of solutes.

2.2.1. Isotonic cell volume The isotonic cell volume varies in a relative narrow range between 20 and 28 ␮m3 in human, primate and large domestic animal species like boar and bull (Petrunkina, 2007). Using both morphometrical and, more recently, electronic cell sizing; stallion sperm volume has been found to fall within this range (24.4 ␮m3 , Pommer et al., 2002). However, as such accurate data has not been available for cryobiological modelling until recently, modelling has often been performed based on morphometric measurements or even on simplified geometrical approximations. These have yielded overestimated values (e.g. stallion sperm were modelled as cylinders with a volume of about 50 ␮m3 ; Devireddy et al., 2002a). The sperm head area in fertile stallions is reported to be about 10–12 ␮m2 , representing only about 10% of the total sperm surface area (estimated as 150 ␮m2 : Gravance et al., 1996; Casey et al., 1997; Gao et al., 1997). Nevertheless, cell size/head surface area was correlated with fertility (Casey et al., 1997). The mean measurements for sperm length, area and perimeter were significantly higher in a subfertile group of stallions; the width of the heads also tended to be larger than those of fertile stallions. This finding is similar to those observed in boar and bull using electronic volume technology (Petrunkina et al., 2001a,b, 2004a; Khalil et al., 2006), although no further studies are yet known regarding variability of cell volume in stallion spermatozoa or presence of subpopulations with different functional characteristics. 2.2.2. Osmotic behaviour and tolerable excursions The intracellular and extracellular aqueous solutions containing electrolytes, osmolytes, macromolecules and gases can exist in different states. During freezing and thawing, due to the phase transitions taking place, the concentration and distribution of these solutes in the extracellular and intracellular environments undergo major changes. These changes can be understood by investigating water and solute transport across the cell membrane. Two assumptions are made: (a) cells are surrounded by a membrane which is impermeable for solute but permeable for water and (b) cells are normally in a state of osmotic equilibrium. According to osmotic theory, as osmotic conditions change, water movement will take place until the osmotic pressure of the cytoplasm matches the external osmotic pressure (Alberts et al., 1990; Pegg, 2002). During cryopreservation, major changes in osmolality take place within and surrounding the sperm cell. Characterization of cell osmotic behaviour includes determining the cell volume excursions in anisotonic solutions containing only impermeable solutes, establishing the range of osmotic tolerance of cells, and

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establishing the range within which, if transferred back to isotonic conditions, cells are capable of returning to their normal cell volume and to maintain motility. The osmotic tolerance of spermatozoa varies between species. Boar and bull spermatozoa are rather sensitive to osmotic changes (maximal cell volume excursion for maintaining motility about 0.97–1.03), whereas broader tolerance limits were observed in murine species (Willoughby et al., 1996; Gilmore et al., 1998; Guthrie et al., 2002). Although stallion spermatozoa are able to return to normal levels of cell volume after incubation in aniso-osmolal solution (75–900 mosmol/kg), only mild changes of osmolality (up to 450 mosmol/kg) allowed a return to normal levels of sperm function; sperm viability, mitochondrial membrane potential and motility were affected by both hypotonic and hypertonic challenges (Pommer et al., 2002). It is important to distinguish between primary osmotic tolerance of spermatozoa (ability to maintain adequate membrane integrity if transferred into aniso-osmotic solutions), and tolerance after exposure to osmotic shock (especially hyperosmolality) and subsequent return to iso-osmotic conditions. The latter, known as post-hypertonic injury, is associated with a greater loss of membrane integrity than primary aniso-osmotic shock, and is very important for cryopreservation. Primary hypotonic stress is often more damaging than an abrupt return to iso-osmolality from tolerable hypoosmolality (e.g. in boar, Gilmore et al., 1996). 2.2.3. Osmotically inactive cell volume At a constant temperature and under equilibrium conditions, cell volume will change reciprocally with osmolality if the cells fulfil the Boyle Van’t Hoff equation:   π0 V = (V0 − Vb ) + Vb πe where V0 is the isotonic volume, π0 is the isotonic osmotic pressure, πe the experimental osmotic pressure and Vb the osmotically inactive volume. Spermatozoa from large mammalian species appear to act over a wide range of osmolalities as perfect osmometers according to the Boyle Van’t Hoff relationship. It is therefore possible experimentally to calculate the osmotically inactive cell volume Vb . This volume is obviously limiting with respect to the cell volume excursions. Its value in stallion sperm (70.7%) appears to be somewhat higher than in other species such as bull and human (Table 2). Stallion sperm follow Boyle Van’t Hoff behaviour over a moderate osmotic range (150–900 mosmol/kg), indicating a lower osmotic tolerance than bovine sperm but comparable with boar and human sperm. 2.2.4. Hydraulic conductivity The Boyle Van’t Hoff equation describes only the equilibrium cell volume without giving any information about how rapidly this equilibrium can be achieved. The kinetics of cell volume as a function of time is determined by the physical structure of the membrane and depends on its hydraulic conductivity Lp (water permeability). Cells with a higher Lp will reach equilibrium faster. The hydraulic conductivity is a property that is unique to a particular cell type. However, even within one cell type, the hydraulic conductivity varies between animal species. Estimates of Lp vary depending upon the accuracy of the measuring technique (e.g. time-to-lysis, stopped flow, or electronic cell sizing). For example, the determination of Lp in bovine spermatozoa using a time-to-lysis technique yielded a Lp value of 10.8 ␮m/(min atm) (Watson et al., 1992), indicating that osmotic equilibrium would be achieved within less than 1 s, whereas determination of Lp in bovine or human spermatozoa by electronic volume measurement or by stopped-flow fluorimetry yielded values of 0.7–1.8 ␮m/(min atm) (Gilmore et al., 1995; Chaveiro et al., 2004), implying

Table 2 Cryobiological determinants of mammalian spermatozoa Bull

Human

Stallion

Iso-osmotic cell volume (␮m3 )

26.3a ; 24.1b

23.5c

28.2d

24.4e

Osmotically inactive cell volume (%)

67a ;

61c

50d

71e

Range of Boyle Van’t Hoff model—behaviour (mosmol/kg)

185–900a ; 150–900b

150–1200c

145–900d

150–900e

Osmotic tolerance

Motility (tolerable volume excursions) Motility recovery (mosmol/kg)

0.97–1.02f ∼280–320h (∼80%)

0.92–1.03c 270–360c (∼90%)

0.75–1.1g 240–600g

Viability recovery (mosmol/kg) Viability (mosmol/kg)

185–900a 185–900a

250–732k

∼90–1100g,j (∼80%) ∼up to 2800j

∼0.89–1.0e 300–450e (∼70%) 200–400i (∼50%) 300–600e 300–900e ; 325–600i

Lp (−CPA* , suprazero) (␮m/min·atm) Lp (−CPA, subzero), (␮m/min·atm)

1.03a ; 0.84f 0.02n

10.8l ; 0.69m 0.036o

1.84d 0.14p

0.02q

Lp (+CPA, suprazero) (␮m/min·atm) Glycerol Dimethylsulfoxide Ethylene glycol

0.14h 0.12h 0.20h

0.29m 0.91m 0.39m

0.77d 0.84d 0.74d

Lp (+CPA, subzero) (␮m/min·atm)

0.005n

0.025o

0.04p

Ps (10−3 cm/min) Glycerol Dimethylsulfoxide Ethylene glycol

0.48h 0.93h 1.98h

1.75m 1.72m 1.49m

2.07d 0.80d 7.94d

* CPA,

63b

0.008q

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Boar

cryoprotective agent.

a Gilmore et al., 1996; b Petrunkina and T¨ opfer-Petersen, 2000; c Guthrie et al., 2002; d Gilmore et al., 1995; e Pommer et al., 2002; f Curry et al., 2000; g Gao et al., 1995; h Gilmore

et al., 1998; i Ball and Vo, 2001; j Gao et al., 1993; k Liu and Foote, 1998; l Watson et al., 1992; m Chaveiro et al., 2004; n Devireddy et al., 2004; o Li et al., 2006; p Devireddy et al., 2000; q Devireddy et al., 2002a.

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that equilibration would take more than 10 s in these species. The most accurate estimations for Lp above 0 ◦ C are usually obtained by cell sizing whereas differential scanning calorimetry is most suitable for measuring subzero permeability (Gilmore et al., 1995; Devireddy et al., 1999, 2002a, 2004; Thirumala et al., 2003). The property of hydraulic conductivity is associated with the ability of cells to survive cryopreservation stress, because it characterizes how quickly the cells can respond to a changing osmotic environment (Mazur, 1984). Although Lp has been determined in a broad range of sperm species (Petrunkina, 2007 and references therein), no measurements have been made for stallion sperm in the absence of extracellular ice using electronic cell sizing. Since freezing and thawing cause very large changes in the osmotic environment of cells, Lp appears to be a crucial parameter in determining cellular response to exposure to deep temperatures. 2.2.5. Permeability coefficients of cryoprotectants In practice, during cryopreservation, the cellular environment cannot be considered as a simple aqueous solution of impermeable solutes. The conservation media contain additional substances like cryoprotectants, which can permeate the cell membrane. In this case, both water and solute flow will take place; moreover, these flows will be coupled. Water flow through the membrane will take place due to the osmotic gradient. However, this osmotic gradient will be changing because the osmotic pressure of the intracellular and extracellular solutions will depend on transport of the permeable solute across the membrane pore (Fuller et al., 2004). For example, if cells are more permeable to water than to a cryoprotectant, they will initially shrink because of water efflux. Subsequently, the cell volume will increase as the cryoprotectant enters the cell. In the generalized form (for k different permeable solutes), these two processes can be defined mathematically using two differential thermodynamic equations known as the Kedem–Katchalski model (Kedem and Katchalski, 1958). If the solution contains only impermeable solutes plus one cryoprotectant, the equations are simplified and can be used for model calculations. To be able to perform these calculations, knowledge of the cryoprotectant permeability coefficient Ps is required as well as the effect of the cryoprotectant on hydraulic conductivity Lp . The results of published studies indicate that there is a broad variation in the membrane permeability of commonly used cryoprotective agents. The permeability of a given agent also varies considerably between sperm species (Table 2). It is worth noticing that no cryoprotectant permeabilities have been measured using modern technologies on stallion spermatozoa. 2.2.6. Reflection coefficient The reflection coefficient σ is the fraction of permeable solute allowed through the membrane pore (i.e. 0 < σ < 1). It is used in three-parametric predictions of volume excursions, although in the majority of cases a two-parameter model for volumetric changes due to water flux V and solute flux Vs , without using the reflection coefficient, is adequate for predicting osmotic response (Kleinhans, 1998; Katkov, 2000, 2002). 2.2.7. Activation energy The activation energy of hydraulic permeability Ea , which is calculated using the Arrhenius equation and an Arrhenius plot for data collected at different temperatures, allows cellular response to be extrapolated to different temperature conditions. 2.2.8. Mathematical modelling of tolerable volume excursions and optimal cooling rates The above parameters, determined by fitting experimental data to the transport equations and Boyle Van’t Hoff and Arrhenius plots, will theoretically determine

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the kinetics of hydration and cell response to the cryopreservation process (Mazur, 1984). However, several discrepancies have already been found between theoretical approximations and experimental observations with respect to maximum cell volume response to aniso-osmolality. Moreover, optimal cooling rates determined empirically by no means agree with those calculated according to two-parameter models, especially in the case of mammalian spermatozoa. Water permeabilities extrapolated to subzero temperatures based on data obtained at suprazero temperatures in the absence of extracellular ice and cryoprotectants lead to dramatic discrepancies between theoretical and experimental optimal cooling rates (Curry, 1994; Gao et al., 1997). Whereas observed optimal cooling rates for most mammalian species lie in the area of ∼50 ◦ C/min (Devireddy et al., 1999, 2004), mathematical calculations of Lp and Ea imply that very high rates (up to 1000 ◦ C/min) are appropriate for dehydration (Curry, 1994). In fact, such high rates will be lethal for spermatozoa: a major decrease in viability has been observed in a range of species after cooling at no more than 100–300 ◦ C/min. For stallion spermatozoa, there was a measurable drop in the post-thaw viability of sperm cooled at 200 ◦ C/min and those cooled at 2 ◦ C/min. This was probably because of the rapid cooling and consequent formation of intracellular ice and slow cooling, respectively (Devireddy et al., 2002a). Optimal cooling rates for stallion sperm are about 29 ◦ C/min in the absence of cryoprotective agents and about 60 ◦ C/min in their presence, as calculated at subzero temperatures (Devireddy et al., 2002a). The complexity of cryoprotectant action may be one of the reasons for the discrepancy between theory and practice of sperm cryobiology. Another reason may be that the presence of permeant solutes changes the water permeability. Indeed, it has been shown in a large number of mammalian sperm species that addition of cryoprotectants such as dimethylsulfoxide, ethylene glycol and glycerol affects the hydraulic conductivity (Table 2). In human, boar and mouse sperm, for example, the addition of cryoprotectants reduces the hydraulic membrane permeability (see Petrunkina, 2007 and references therein). In stallion sperm, a similar effect has been observed in the presence of extracellular ice (Devireddy et al., 2002a). The degree of effect varies both between types of cryoprotectants and between animal species. Finally, it has been shown recently that the values of membrane parameters (hydraulic permeability Lp and activation energy Ea ) of mammalian spermatozoa measured at subzero temperatures in the presence of extracellular ice differ considerably from those measured at suprazero temperatures (Devireddy et al., 1999, 2000, 2002a,b, 2004). Indeed, it has been reported that hydraulic permeability at subzero temperatures is lower than at suprazero temperature by one order of magnitude, and that activation energy at subzero temperatures is higher by a factor of 2 than at suprazero temperatures. Such divergence may be related to cooling-induced changes in the sperm plasma membrane structure such as lipid phase transition, and/or chilling injury (temperature-dependent alterations in macromolecular structures). In stallion spermatozoa, cold shock resulted in a significant drop in the hydraulic membrane permeability (Devireddy et al., 2002b). Cold shock is believed to involve phase transition in the sperm membrane lipids and is linked to changes of membrane permeability to major ions (Parks and Lynch, 1992). Taking into account these divergences between supra- and subzero permeabilities and activation energies, theoretically calculated optimal rates can be brought into better agreement with experimentally determined ones. It is clear that further progress in improving sperm cryopreservation technology will depend on more accurate estimates of cell size and more exact determination of membrane water permeability parameters in the presence of cryoprotectant at subzero temperatures using differential scanning calorimetry. Unfortunately, it is not possible to measure the permeability of cryoprotective agents at subzero temperatures by this method.

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3. Biological complexity of spermatozoa One of the main reasons for the discrepancy between the theoretical and experimental behaviour of spermatozoa during the freezing/thawing process is their biological complexity. Most calculations used for the estimation of optimal cooling protocols are based on the theory of ideal solutions, considering the spermatozoon as a symmetrically spherical- or cylinder-shaped semi-permeable ‘bag with salt water’. However, such an approximation is not even close to the reality. Firstly, the sperm cell plasma membrane is not a simple semi-permeable barrier. It is a complex dynamic structure composed of lipids and phospholipids distributed as a bilayer by metabolic activity. Within the membrane are embedded proteins and glycoproteins with access to the external environment as well as to the interior. One class of such proteins, aquaporins, is involved in water transport. Others function as ion channels with specificity to particular ion species; some of the channels are energy-requiring pumps which transport the ions against concentration gradients. The physical and chemical structure of the plasma membrane and its associated metabolic activity is complex and affected by temperature: changes in molecular structure at deeper temperatures will alter solute and water transport through pores and channels, and changes in mobility and distribution of lipids will affect diffusion through the lipid bilayer (cf. James et al., 1999). Secondly, the interior of the spermatozoon is not composed simply of water with dissolved ions. Within the cell are membrane-bounded compartments such as mitochondria and the acrosome, whose functional activities are likely sensitive to cryopreservation conditions, even if their membranes remain physically intact. The cytoplasm contains many proteins which interact with each other and with transmembrane proteins in subtle ways to provide means of modulating cellular activity in response to changes in the sperm’s environment (signalling pathways; Alberts et al., 1990; Fuller et al., 2004). Exposure to deep temperatures can affect this interaction and thereby critical signalling mechanisms; there is already evidence that some sperm intracellular signalling pathways can be affected during cooling and cryopreservation (Green and Watson, 2001; Cormier and Bailey, 2003; Petrunkina et al., 2005a; Thomas et al., 2006). Finally, long-term cell osmotic response is much more complex than described by the Boyle Van’t Hoff relationship. There is a body of evidence that cells are able to moderate or reverse osmotically induced swelling or shrinking by activation of ion channels and/or transport mechanisms. In spermatozoa particularly, osmotically induced shrinking or swelling is known to activate ion channels, either by changes in phosphorylation states or by modification of the cytoskeleton, whence major intracellular ions are allowed to leave or to enter the cell and subsequent coupled water transport takes place (Petrunkina et al., 2004b,c, 2005b, 2007a,b). Such control of cell volume involves finite processes which become activated within a time range of a few seconds to several minutes in mammalian spermatozoa (Petrunkina et al., 2007a). It is worth noting that control of sperm cell volume seems to be associated with fertility; inability to maintain a stable isotonic cell volume indicates poor functional ability of the ejaculate (Yeung et al., 2000; Petrunkina et al., 2001a, 2004a; Khalil et al., 2006). The impact of volume control mechanisms on cryopreservation response in stallion remains to be elucidated. Apart from lethal changes produced by cryoinjury in the general way through intracellular ice formation or solution effects, there may be sub-lethal changes in spermatozoa at a molecular level which affect the processes specific to their fertilizing function. After deposition in the female genital tract, the spermatozoa must first bind to regions in the oviduct which act as storage areas for the spermatozoa to await the time of ovulation. At ovulation, the bound sperm begin to undergo the changes collectively known as capacitation which enable them to acquire the ability to interact with the egg (i.e. undergo an egg-induced acrosome reaction and fusion with the

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oocyte). These changes are thought to be initiated by an increase in the bicarbonate/CO2 content of the oviduct environment. This activates a cAMP-dependent PKA signalling pathway that brings about a remodelling of the sperm plasma membrane to enable major removal of cholesterol by binding proteins (Harrison and Gadella, 2005). Further subsequent modifications take place, such as modulation of surface binding sites, protein tyrosine phosphorylation, increased internal pH and Ca2+ levels and alterations in membrane polarization (Neild et al., 2005). There are indications that stallion spermatozoa may undergo capacitation events similar to those in other large animals, including modification of plasma membrane structure, tyrosine phosphorylation, and low-level production of the superoxide anion (Rathi et al., 2001; Pommer et al., 2003; Neild et al., 2005; Burnaugh et al., 2007). Detailed understanding of the capacitation process must play a crucial role in improving semen cryopreservation and in explaining its detrimental effects on cell function. Although sperm are subjected to cryopreservation soon after ejaculation, and therefore before the onset of capacitation, the physiological characteristics of thawed sperm show similarities with those of advanced capacitation but are not absolutely identical at the molecular level (Watson, 1995). This phenomenon is currently known as ‘cryocapacitation’ and it has been observed in spermatozoa of large animals, particularly in stallion sperm, where the characteristics of capacitation were modified following cryopreservation (Green and Watson, 2001; Thomas et al., 2006). Frozen–thawed sperm may be more sensitive to inducers of capacitation. This could explain their limited life span when compared with fresh sperm. Also, cryopreserved stallion sperm show lower ability to bind to the epithelium in the distal portion of the oviductal isthmus (Dobrinski et al., 1995). In stallion, as in other large domestic animal species, only viable, motile, uncapacitated, morphologically normal sperm bind at this site. Such binding leads to suppression of capacitation-related events such as influx of calcium ions into the cell and tyrosine phosphorylation of sperm proteins (Thomas et al., 1994; Dobrinski et al., 1996); the sperm are released and begin to undergo capacitation around the time of ovulation, perhaps following changes in the bicarbonate/CO2 content of the oviductal secretions. To provide a sufficient number of fertilization-competent sperm population at the time of ovulation, a sufficient number of sperm should be able to bind to the oviductal epithelium and capacitation should not be completed until ovulation occurs. If cryopreserved spermatozoa initiate capacitation or the acrosome reaction prior to insemination, due to advanced membrane destabilization, this would likely result in a diminished capacity to interact with the female genital tract environment and subsequently to penetrate the zona pellucida and fertilize the egg (cf. Petrunkina et al., 2007b and references therein). However, although the mechanisms of capacitation have been studied extensively in other domestic animal species like bull and boar, there is a lack of similar studies with respect to stallion sperm. Major signalling mechanisms have not been identified, only coarse membrane changes have been characterized with molecular markers, and even the physiological acrosome reaction has not been studied satisfactorily. To understand the mechanisms by which cryopreservation alters stallion sperm fertilizing function, much better knowledge regarding the processes of capacitation in this species is required. 4. Reflections with respect to improving cryopreservation techniques In many animal systems, only small improvements have been made in the basic techniques of sperm cryopreservation since the early 1950s (Woods et al., 2004). Whereas in the cattle breeding industry considerable successes have been achieved in both semen freezing and associated insemination technology, application of these advances to horse breeding has been relatively

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unsuccessful. It appears that transfer of optimal freezing protocols from one cell type to another cell type, and from one species to another, is often unsuccessful. Although this wide biological variability may partly be due to differences between species in sperm physiology and biochemistry, and to the physiology and anatomy of the female genital tract (Holt, 2000), poor understanding of the fundamentals of the cryobiological processes themselves may play a large part in limiting progress. Historically, there have been two almost independent approaches to advancing knowledge in the field of cryopreservation. The approach that has been followed to a great extent in animal reproductive technology, has been almost entirely empirical and has usually missed out detailed quantitative analysis while concentrating on cell physiological characteristics. The alternative biophysical approach using data and knowledge of cryobiological determinants to predict optimal freezing protocols has been applied but has shown obvious limitations in its applicability to living cellular systems. A critical example of this latter problem is the evident discrepancy between theoretically predicted optimal cooling rates and the empirically obtained cooling rates that in reality result in maximal cell survival (Gilmore et al., 2000). Naturally, these limitations have prevented general acceptance of the obvious potential of this latter approach. Nevertheless, continuing studies into the biophysics of cryopreservation shows that it is crucial to obtain accurate information regarding the relevant determinants. It has become clear that the quantitative values associated with these vary with the conditions under which they are being measured. While the primary cryobiological parameters (isotonic volume, osmotically inactive volume, range of osmotic tolerance, tolerable volume excursions, subzero hydraulic permeability) have been determined, others such as suprazero hydraulic permeability in the presence and absence of cryoprotectants, or cryoprotectant permeabilities, are still unknown. Thus, further biophysical studies on stallion sperm need to be performed, using modern technologies of electronic cell sizing and differential scanning calorimetry, to allow accurate quantitative determination of relevant cryobiological parameters and thence valid mathematical modelling. Finally, owing to the biological complexity of the sperm cell, the physiological aspects of cryopreservation need to be taken into account. Further studies on stallion sperm are required to understand how and why cryopreservation alters sperm functions and in what way the process may influence the selection by the female tract of cells with higher fertilizing potential. Of particular importance will be characterization of the mechanisms and dynamics of sperm volume regulation, sperm–oviduct interaction, capacitation and cellular signalling. Conflicts of interest None. References Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D., 1990. Molekularbiologie der Zelle. VCH, Weinheim. Alvarenga, M.A., Papa, F.O., Landim-Alvarenga, F.C., Medeiros, A.S.L., 2005. Amides as cryoprotectant for freezing stallion semen: a review. Anim. Reprod. Sci. 89, 105–113. Ball, B.A., Vo, A., 2001. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J. Androl. 22, 1061–1069. Burnaugh, L., Sabeur, K., Ball, B.A., 2007. Generation of superoxide anion by equine spermatozoa as detected by dihydroethidium. Theriogenology 67, 580–589. Casey, P.J., Gravance, C.G., Davis, R.O., Chabot, D.D., Liu, I.K.M., 1997. Morphometric differences in sperm head dimensions of fertile and subfertile stallions. Theriogenology 47, 575–582.

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