Fundamentals and recent development in cryopreservation of bull and boar semen

Abstract

pes of lipid will aggregate in domains of gel-like (` frozen') lipid, thus excluding other lipid species that still remain in the liquid-crystalline (melted) state. Membrane proteins are also excluded from these gel domains and consequently find themselves in a non-physiological lipid environment (lateral phase separation (7,22). This is believed to impair the function of membrane proteins that are necessary for structural integrity (cytoskeleton) or ion metabolism (ion pumps). A second change in the environment of the sperm takes place when liquid water is converted into ice. Spontaneous ice nucleation will usually occur after the solution is supercooled to a temperature between -Sand -15 °C. This is due to the fact that small ice crystals have a large surface tension, which makes them thermodynamically unstable. Therefore, spontaneous ice nucleation is a random process, occurring only when, by chance, enough molecules with a lower-than-average kinetic energy get together and form a stable ice nucleus. Once this has happened, the ice crystals will grow rapidly in all directions. The release of the latent heat of fusion then causes the sample to warm up abruptly, until the freezing/ melting temperature of the solution (of the remaining unfrozen fraction) is reached. At this point, ice formation stops or proceeds at a rate governed by the rate at which the sample is further cooled. What remains is the so-called unfrozen fraction, in which all cells and all solutes are confined. The concentration of the sugars, salts, and cryoprotectant (e.g. glycerol), and, consequently, also the osmotic pressure of the unfrozen fraction, increases rapidly. At the same time, the volume of the unfrozen fraction decreases rapidly. The increase in the osmotic strength causes an efflux of water from the cells. Therefore, the intracellular concentration of salts and glycerol also increases until the osmotic pressure inside the cells is as high as that outside the cells. As cooling continues, these processes continue until the viscosity of the unfrozen fraction becomes too high for any further crystallization. All the above physical and physicochemical changes imply as many hazards for the cells:-Dehydration of the membranes and intracellular components, with subsequent loss of stability of the lipid bilayer, and denaturation of proteins (3, 14, 27). -The efflux of water itself is proposed to be a cause of damage to the cell membrane (19, 20). -The fast efflux of water causes a rapid decrease in the volume of the cells to some 50 % of their original volume, which leads to structural deformation of the cells. The cells find themselves confined to very narrow channels of unfrozen solution, squeezed in between growing masses of ice, which could also lead to mechanical stress in the cells (24). Extremely high concentrations of salts could be a cause of damage by itself (15), affecting the cell membrane or intracellular components. Also, extracellular salts could leak into the cells, changing the intracellular ion composition FUNDAMENTALS A...

Effect of equilibration times, freezing, and thawing rates on post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa

“…Similarly, Sukhato et al (2001) reported that automated freezing at the rate of À20 or À30°C/min yielded better sperm PM in buffalo compared to automated freezing at the rate of À10°C/min. It is, therefore, suggested that if the freezing rate is within the required values (50-100°C/min), this results in less excessive intracellular dehydration, less excessive intracellular solute concentrations, and less shrinkage of the cells (Mazur, 1984;Woelders, 1997). Moreover, at optimum freezing rates, the spermatozoa remain exposed to the unfavorable conditions for a shorter period of time (Woelders, 1997).…”

Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa

“…For example, an L p value of 0.5 mm/min-atm and an activation energy of 3 kcal/mol results in a theoretically predicted optimal rate of freezing bovine sperm of ,5000 8C/min, when, in fact, experiments show that the 'optimal cooling rate' for bovine spermatozoa is 30 -100 8C/min, with a major decrease in viability after cooling at 300 8C/min (Rodriguez et al 1975, Robbins et al 1976, Foote & Parks 1993, Woelders 1997, Woelders & Malva 1998, Kumar et al 2003. One explanation of this discrepancy is that the values of water transport parameters at subzero temperatures in the presence of extracellular ice are markedly different than those reported in the literature at suprazero temperatures.…”

“…Today, the worldwide cattle industry is based on artificial insemination and frozen semen, and cryopreservation has allowed exploitation of superior sires and achieved rapid, large-scale genetic improvement in cattle stocks coupled with a reduction in disease transmission (Foote 1999). This impact would not have been possible without successful freezing of bull spermatozoa (Parkinson & Whitfield 1987, Foote & Parks 1993, Woelders 1997, Vishwanath & Shannon 2000; also see Foote 2002 for a historical review on artificial insemination).…”

Effect of Cryopreservation on Sperm Quality and Fertility

“…In response to this newly developed osmotic pressure gradient and the fact that water within the spermatozoon is slower to form ice crystals than the water in the surrounding medium, water passes out of the spermatozoa, particularly from the spermatozoon head, across the semi-permeable plasma membrane. Consequently, the spermatozoon becomes increasingly dehydrated (Andrabi, 2007;Watson, 2000;Woelders, 1997). The rate of efflux of water from the spermatozoa also depends upon the speed of temperature drop: the slower the drop, the greater would be the time needed for the efflux of water, and hence a much greater dehydration.…”

“…A successful cryopreservation should, therefore, aim at arriving at an optimum cooling rate that will provide a compromise between all these factors. There are two main temperature ranges of concern regarding damage to spermatozoa during freezing: the period of supercooling (0°C to -5°C) and the formation of ice crystals (-6°C to -15°C) (Woelders, 1997). Excessive supercooling results in a rapid ice formation, with the possibility of physical damage.…”