Mechanisms of cell injury during cryopreservation

Semen cryopreservation techniques have been in practice for the past 50 years and allow long-term storage of semen and hence its virtually unlimited availability. This facilitates the large-scale provision and dissemination of highly valuable genetic material at will. Cryopreservation of semen involves the freezing of spermatozoa to –196 °C, the boiling point of liquid nitrogen, a commonly used medium for freezing and storage purpose. This increases the viability of the individual cells by slowing down their metabolic rate, thereby reducing the rate at which substrates are used and toxins are produced. Semen may also be stored after cooling to 5–8 °C and will survive for 24–48 hours without a significant decline in motility, and even up to 96 hours without a significant drop in fertilization rates. Although this may provide an efficient and successful means of short-term storage, it has some adverse effects on the spermatozoa manifested as a decline in viability rate, structural integrity, motility, and conception rates.^5,6 Cryopreservation, on the other hand, offers the option of indefinite storage of semen in liquid nitrogen with acceptable post-thaw fertility rates. Cryopreservation can be detrimental to sperm function and fertility even with the latest advances in techniques. However, it is not the long-term storage of cells at these temperatures that is damaging, but rather the progression to these temperatures and back to normothermia which results in cryoinjury.⁷ Thus the changes that occur during freezing are mainly ultrastructural, biochemical, and functional. These can impair sperm transport and survival in the female reproductive tract and reduce fertility in domestic species.⁸

The effects of cryopreservation on function and viability have been extensively studied for bovine sperm. Spermatozoa have a very limited biosynthetic activity of their own and depend mostly on catabolic processes to function.⁹ Thus to halt these metabolic processes the cells need to be cooled below –130 °C. Sperm, like other cells in the body, are composed of various organelles containing water, which can form intracellular and extracellular ice crystals during the freezing process. At temperatures around –5 °C the intracellular and extracellular water remains unfrozen in a supercooled metastable state. However, between –5 and –10 °C, ice forms in the extracellular medium, while intracellular water remains supercooled. At this point, the rate of cooling must be slow enough to permit cellular dehydration to occur, avoiding the freezing of the intracellular water yet fast enough to avoid exposing the cell to a hyperosmotic condition subsequent to dehydration. Severe dehydration leads to solution-effect injury caused by denaturation of macromolecules and extreme shrinkage of the cell up to irreversible membrane collapse.¹⁰ Another damaging effect is the mechanical stress of ice formation all around the cell, which will be constrained to a very limited space of unfrozen solutes.¹⁰ Cell viability plotted as a function of rate of freezing presents an inverted U-shaped curve. Primary causes for cellular damage are largely dependent on the cooling rate employed during the freezing process. The most appropriate freezing rate is the fastest one that allows freezing of extracellular water without intracellular ice formation. Fast cooling between 30 °C and 0 °C results in cell injury in some sperm cells, called “cold shock,” with deleterious effects on the cytoskeleton and genome-related structures and causing cytoplasmic fracture.¹¹ Cold shock also alters permeability of various membrane structures on the plasma membrane, mitochondria, and acrosome. During cryopreservation, ice crystals form in the extracellular medium, increasing the osmolality of the unfrozen water. Because of this difference in the osmotic gradient, the intracellular water diffuses out of the sperm thus dehydrating the cell and the plasma membrane. At thawing, this phenomenon is repeated in reverse order as extracellular ice crystals melt and water starts diffusing in and rehydrating the cell. Because of such drastic changes in the volume and osmotic stress, there can be irreversible damage and ultrastructural deformation of the plasma membrane.

Sperm membranes are composed of many phospholipids (depending on species), with each phospholipid having a precise phase transition temperature. The degree of structural damage to these membranes depends on the temperature and the lipid composition of the membrane.¹² The characteristics of membranes that affect their sensitivity include cholesterol/phospholipid ratio, content of nonbilayer-preferring lipids, degree of hydrocarbon chain saturation, and protein/phospholipid ratio.¹ Some lipids aggregate in domains of gel-like (frozen) lipid, thus excluding other lipid types that remain in the liquid crystalline (melted) state.^1,13 This ultimately leads to phase separation in which membrane proteins can become irreversibly clustered, leading to loss of function.¹⁴ The specific phase transition temperatures for the different phospholipids in the membrane result in lateral migration with rearrangement of membrane components and lipid phase separations within the plane of the membrane. The lateral migration may create microdomains of nonbilayer-forming lipids and may modify protein surrounding environments. On thawing, these alterations predispose apposing membranes to fuse and affect protein activity, leading to overall altered membrane permeability to water and solutes.¹ This loss of membrane permeability also interferes with ion pumps and results in influx of Ca²⁺ into the sperm cell as well as loss of membrane ATPase activity. Damage to the Ca²⁺ regulatory systems by freezing and thawing may predispose spermatozoa to inaccurate timing of capacitation and the acrosome reaction, contributing to reduced fertilizing capacity of cryopreserved bull semen.¹⁵

Role of extenders used for cryopreservation

Extenders or diluents are routinely added to protect sperm during liquid storage or cryopreservation. Regardless of species, the role of semen extenders is to (i) provide nutrients as an energy source; (ii) buffer against harmful changes in pH; (iii) ensure appropriate physiologic osmotic pressure and concentration of electrolytes; (iv) prevent growth of bacteria; (v) protect from cold shock during cooling; and (vi) have cryoprotectant(s) to reduce the amount of freezing damage.¹⁶ Over the past 65 years, the cryoprotective media for sperm storage have been continuously revised but the basic ingredients remain unchanged, with egg yolk and/or milk and glycerol representing the indispensable compounds of practically all media used for bull sperm preservation in the liquid or frozen states. Extenders used for diluting bull semen typically are egg yolk and/or skimmed milk-citrate/Tris-based buffers with added simple sugars and antibiotics, with or without cryoprotectants depending on whether they are one-step or two-step extenders. These components provide an acceptable buffering capacity, osmolality and energy in the form of metabolizable substrates, minimize bacterial growth, and also protect sperm from decreases in temperature.

As mentioned earlier, sperm can suffer cold shock as a result of a sudden reduction in temperature, which causes structural and biochemical damage. This can be prevented by cooling semen slowly in the presence of protective agents. Phillips¹⁷ first reported the value of adding egg yolk to bovine semen to afford such protection. Subsequent studies have shown that egg yolk not only increases the fertilizing ability of spermatozoa at ambient temperatures but also appears to prevent sperm cell damage during cooling and freezing.^18–21 Over the years the concentration of egg yolk added to extenders has been reduced from 1 : 1 (volume/volume) to 20–25% of volume as this has been shown to improve sperm survival.^22,23 The low-density lipoproteins (LDLs) in yolk have been shown to play an important role in protecting sperm, with some studies reporting that LDL by itself is better than whole egg yolk in preserving sperm motility after freezing.^24,25 Besides egg yolk, skimmed milk has also been found to be very efficient in protecting sperm during semen storage at 4 °C or in cryopreservation,^26–28 and has the same composition as whole milk but contains less than 0.1% lipids (mostly triglycerides). The protective constituent of skimmed milk is not the lipid fraction but rather the protein constituents known as casein micelles. There are various hypotheses on how LDLs provide protection. It has been suggested that the phospholipid fraction of LDL protects sperm by forming a protective film on the sperm surface²⁹ or by replacing sperm membrane phospholipids that are lost or damaged during the cryopreservation process.^30,31 Vishwanath et al.³² suggested that egg yolk lipoproteins compete with detrimental seminal plasma cationic peptides (<5 kDa) in binding to the sperm membrane and thus protect the sperm. However, recent studies and research favors the idea that LDL interacts with the major proteins of bull seminal plasma and this interaction appears to be crucial for sperm protection.³³ A family of major proteins, known as binder of sperm (BSP), found in seminal plasma binds to sperm at ejaculation and modifies the sperm membrane by removing cholesterol and phospholipids. This may adversely affect the ability of sperm to be preserved. LDL from egg yolk and casein micelles and whey proteins in skimmed milk sequester these BSPs, thereby maintaining sperm motility and viability during storage.³⁴

Apart from egg yolk and skimmed milk, extenders used for diluting bovine semen also contain various buffers, with phosphate being one of the earliest to be used. However, sodium citrate has largely replaced phosphate due to its superior ability to promote sperm survival at 5 °C. Citrate also improves the solubility of proteins fractions in the egg yolk due to its chelating properties. Many zwitterionic buffers such as Tris [tris(hydroxymethyl)aminomethane], TES [N-tris(hydroxymethyl)-methyl-2-aminoethane sulfonic acid], and Tris titrated with TES (TEST) have also been developed and tested over a wide pH range and have proved comparable or superior to citrate buffers. Of these, Tris-based diluents combined with egg yolk have been tested extensively and are used universally for extending bovine semen. In addition, protein components of skimmed milk extenders have also been thought to provide buffering capacity to semen diluents. Because of the potential risk of xenobiotic contamination, research has also started focusing on animal product-free extenders such as coconut and soy milk with satisfactory results. A recent study comparing soy milk tris extender (SMT) with an egg yolk tris (EYT) extender showed no significant differences between sperm in EYT extender and SMT extender with regard to post-thaw motility, viability, membrane integrity, acrosome integrity, and cryocapacitation.³⁵

Spermatozoa require energy for motility and are capable of both aerobic and anaerobic metabolism.³⁶ Most diluents provide an energy source in the form of simple sugars such as lactose, mannose, fructose, and arabinose. Sugars also add osmotic pressure to the medium and act as cryoprotectants. The main effect of sugars and polyols such as glycerol is their ability to replace the water molecule in the normally hydrated polar groups, which helps to stabilize the sperm plasma membrane during transition through the critical temperature zones.¹³ Sugars, like glycerol, also increase the viscosity of the diluent and prevent the eutectic crystallization of solutes increasing the glass-forming tendency of the medium, a property used increasingly in vitrification media.³⁷

Most commercial bovine semen extenders also contain antibiotics to reduce the rate of bacterial overgrowth and its subsequent deleterious effect on semen quality. Bacteria are present in the genitalia and reproductive tract of bulls regardless of fertility status and can be difficult to screen even with the most hygienic and sanitary collection procedures and processing. Moreover, the presence of egg yolk and other extending media provide a good nutrient environment for the growth of these organisms. Historically, penicillin and streptomycin have been the preferred antibiotics used in combination with bovine semen extenders as they are relatively harmless to sperm and, when combined, inhibit a broad spectrum of microorganisms. However, these antibiotics fail to control growth of organisms such as Campylobacter fetus subsp. venerealis and have questionable efficacy against Mycoplasma and Ureaplasma spp. which can survive the freezing process. Continued use of these antibiotics has also led to development of bacterial resistance. Because of these drawbacks the current international standards for semen extenders favor protocols that include treatment of semen and extender with the antibiotics gentamicin, tylosin, lincomycin, and spectinomycin (GTLS), as they are more effective in controlling Mycoplasma, Ureaplasma, Campylobacter fetus, Haemophilus somnus and Pseudomonas in bovine semen.^38,39 Recent studies have shown that bacterial presence in semen can adversely affect the DNA integrity of sperm, and the rate at which this damage takes place correlates positively with the initial bacterial load and bacterial growth rate.⁴⁰ Longevity of DNA is also adversely affected in the presence of GTLS combination. Use of the quinolone class of antibiotics has been shown to increase sperm DNA longevity in semen samples containing bacteria and thus could be of interest in terms of promoting alternative cryopreservation strategies to increase reproductive outcome.⁴¹