Penetrating Cryoprotectants Across Cell Membranes
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Cryopreservation of gametes is important because it would allow us to support a genome resource bank for a breed for an indefinite period of time. Cryopreservation as a technique of storage of goat sperm has advantages but freezing and thawing induces detrimental effects in terms of sperm ultrastructural, biochemical and functional damage,  P.F. Watson, The causes of reduced fertility with cryopreserved semen, Anim Reprod Sci 60-61 (2000), pp. 481-492. Article | PDF (155 K) | View Record in Scopus | Cited By in Scopus (227)resulting in a reduction of motility, membrane integrity and fertilizing ability (Purdy, 2006).
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A cryoprotectant is included in a cryopreservation medium to minimize the physical and chemical stresses resulting from the cooling, freezing, and thawing of sperm cells (Purdy, 2006). Cryoprotectants are normally categorized into two broad classes based on their ability to diffuse across cell membranes, which are penetrating cryprotectant or non-penetrating cryoprotectant (Muldrew, 1998). The colligative action of both penetrating and non-penetrating cryoprotectant allows the cell to survive the condition for a reduction of cell water content during cooling thereby reducing the amount of intracellular freezing (Mc Gann, 1978). This can be shown in the combination of glycerol, dimethyl sulfoxide (DMSO) and dextran (Kundu et al., 2002).
Penetrating cryoprotectants are able to move across cell membranes (Muldrew, 1998) and create the environment for a reduction of cell water content at temperatures sufficiently low to reduce the damaging effect of the concentrated solutes on the cells (Mc Gann, 1978). This is because the penetrating cryoprotectants force the water to be drawn out osmotically from the cell such as sperm cell. As the sperm cell now has less intracellular water, less intracellular ice formation will occur when the freezing point of
the cell is decreased. This is beneficial as intracellular ice can result in cell death and consequently reduced fertility of the semen. The penetrating cryoprotectant also cause membrane lipid and protein rearrangement, which results in increased membrane fluidity, greater dehydration at lower temperatures and therefore an increased ability to survive for cryopreservation processes (Holt, 2000). Penetrating cryoprotectants are solvents that dissolve sugars and salts in the cryopreservation medium (Purdy, 2006).
There are many penetrating cryoprotectants such as glycerol, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol (Leboeuf et al., 2000) and dimethyformamide (Meryman, 1971).
Glycerol was the first Cryoprotectant Agent (CPA) that gained widespread use in cryobiology for red blood cell and sperm cell (Best, 2008) and is the most commonly used penetrating cryoprotectant (Purdy, 2006). However, glycerol has a weakness with its slow movement across those membranes which are permeable to it (Meryman, 1971). Therefore, large number of tissues or cells will appear to be virtually impermeable. Generally about 1 to 8 percent glycerol is used and it showed a great recovery of sperm post-thawing (Singh et al., 1995).
On the other hand, DMSO shows good attributes in the penetration speed. Ehtylene glycol functions as an automobile anti-freeze while propylene glycol was at first used to reduce formation of ice in ice cream also shows to be an effective cryoprotectant (Best, 2008). Formamide need to be assisted by other cryoprotectants in order to vitrify. High concentration of penetrating cryoprotectants use for the purpose of cells protection such as DMSO, ethylene glycol and dimethyformamide can cause toxicity (Meryman, 1971). The more in the number of methyl group (R-CH3), the stronger is the cryoprotectants (Best, 2008).
2.2.2 Non-penetrating Cryoprotectants
Non-penetrating cryoprotectants cannot move across cell membranes (Muldrew, 1998), therefore it osmotically “squeeze” water from the cells primarily during the initial phases of freezing at temperatures between âˆ’10 and âˆ’20 Â°C when these cryoportectant become concentrated in the extracellular regions (Mc Gann, 1978). The high sub-freezing temperatures will cause the non-penetration cryoprotectants dehydrate the cell and allow a rapid cooling instead of slow cooling. This is because slow cooling can lead to extensive damage on the cell (Muldrew, 1998). Good non-penetrating cryoprotectants also shall have the reversible ability in osmolarity although under osmotic stress (Meryman, 1971). Non-penetrating cryoprotectants are generally comprised from polymers that can form extensive hydrogen bonds with water (Muldrew, 1998).
Egg yolk is a common component of most semen cryopreservation extenders for domestic animals (Tuli and Holtz, 1994). Variation from 5 to 50 percent amount of egg yolk used appear to give good results but 20 percent or less are more common concentration of egg yolk used today (Campbell et al., 2003). It has been shown to have a beneficial effect on sperm cryopreservation. This is because egg yolk serves as a protectant of the plasma membrane and acrosome against temperature-related injury as egg yolk and glycerol-free medium used as cryoprotectant showed a positive result of motility after post-thawing the goat sperms (Kundu et al., 2001). Egg yolk was found to be the main cryoprotective agent, but there was a synergistic effect between glycerol (penetrating cryoprotectant) and egg yolk (non-penetrating cryoprotectant) in providing the greatest post-thaw survival (40%) of sperm cells (Pace and Graham, 1974).
Apart from egg yolk, there are some common non-penetrating cryoportectants used which are dextran (Kundu et al., 2002), hydroxyl-ethyl-starch (HES), polyvinyl pyrrolidone (PVP) and polyethylene oxide (PEO) (Muldrew, 1998). Dextran is biphasic (two-phase) as the concentration of dextran is increased, the recovery of sperm motility also increased and reached an optimum value. Any further increases in dextran concentration will result and adverse effect the recovery of motility (Kundu et al., 2002).
Artificial insemination (AI) involves removing semen from a male and introducing it into the reproductive tract of a female, by means of a catheter, at a suitable stage in her reproductive cycle (Stephen, 2007). AI was the first biotechnology applied to improve reproduction and genetics of farm animals (Amer et al., 2008).
According to Leboeuf et al. (2000), AI has an important role in goat breeding, especially in intensive systems of production, to control reproduction and, in conjunction with accurate progeny testing, to improve the production of milk, hair and meat. AI includes more efficient genetic selection schemes and the manipulation and storage of the genetic material.
The semen collected will be subjected to semen evaluation before insemination process is taken. Does can be inseminated with either fresh semen or commercially available frozen semen (Allison and Hagevort, 2009). Artificial insemination remains the main vehicle for the rapid dissemination of valuable genes and the method of choice for the dairy farmer worldwide to improve the genetic quality of their livestock (Vishwanath and Shannon, 2000). The success of an AI program depends on the proper management of semen collection, storage and use (Leboeuf et al., 2000).
Egg is one of the best and most inexpensive sources of high quality protein and contains a balanced distribution of various minerals and vitamins. An egg contains yolk, vitalline membrane, chalazae, albumen, air cell, inner and outer membranes and shell (Figure 2.1). The cholesterol content of eggs is influenced by genetic factors, dietary composition, lay intensity, layer age and medical treatment (Vorlova et al., 2001).
Figure 2.1: Cross-section of a newly laid egg.
Egg yolk is one of the common components of most semen cryopreservation extenders for domestic animals. Chicken egg yolk consistently improved the survival and the maintenance of the fertilizing ability of spermatozoa when included in the conservation medium (Trimeche et al., 1997). The fatty substances of the yolk are mostly triglycerides (true fat) 65.5%, phospholipids 28.3%, and cholesterol 5.2% (USDA, 2000). Egg yolk contains phospholipids, cholesterol and low density lipoproteins which are the factors that provide protection to sperm against cold shock during the freeze-thaw process (KulaksÄ±z et al., 2010). The best quality gametes after cryopreservation are enriched in lipid, triacylglycerol and phospholipid (Cerolini et al., 2001).
Phospholipids contain a glycerol molecule bonded to two fatty acids and a phosphate-containing polar head group (Figure 2.2). Phospholipids are amphipathic as it contains both polar (hydrophilic) and nonpolar (hydrophobic) portions. The hydrophobic portion consists of two fatty acids whereas the hydrophilic portion is the polar head group. This structure allows phospholipids to be a major component of cell membranes (McGuire and Beerman, 2007). Saturated fatty acids have no double bonds between carbon atoms and examples of saturated fat are palmitic acid and stearic acid. Unsaturate fatty acids have one or more double bonds between carbon atoms (-C=C-) which cause kinks in the chain such as oleic acid and linoleic acid (Lee and Arunasalam, 2005). The major fatty acids of avian species egg yolk are oleic acid, palmitic acid, linoleic acid and stearic acid (Choi et al., 2001).
Figure 2.2: A phospholipid molecule.
Cell membrane consists of bilayer of phospholipids with the hydrophilic polar head pointing to the extra- and intracellular spaces that provide stable barrier for the cell (McGuire and Beerman, 2007). According to Lee and Arunasalam (2005), cell membrane is dynamic, fluid structures. The fluidity of the cell membranes can be influenced by the movement of phospholipids, types of fatty acids in the membranes, cholesterol and temperature. Phospholipids molecules can move laterally or change places (flip-flop) and thus give the membrane fluidity. Lateral movement happened rapidly where as flip-flopping across the membrane occurs at a very slow rate.
According to Mazur (1984), membranes are destabilized by several factors which are the passage to and from the storage temperature (thermal stress), the large volume changes associated with water and cryoprotectant movement and the exposure to high salt concentrations (osmotic stress). At a molecular level, changes in membrane organization such as modifications of specific lipid-protein interaction, phospholipids asymmetry and lipid composition are the implication in the loss of cell membrane permeability such as spermatozoa. The reversibility of these changes could be dependent on membrane dynamics and the physical properties of the membranes have been assumed to be one of the pivotal factors in the resistance of spermatozoa to cryopreservation (Hammerstedt et al., 1990). Sperm plasma membrane is a key organelle in controlling sperm cryosurvival (Giraud et al., 2000).
According to Cerolini et al. (2001), polyunsaturated fatty acids in frozen-thawed boar spermatozoa decreased because relative increase in the proportions of saturated owing to the stress of cryopreservation as sperm cell membrane may damage. This is because unsaturated fatty acids have double bonds which form kinks to prevent close packing of the phospholipids molecules, increase fluidity and promote movement of substances across the membrane (Lee and Arunasalam, 2005) (Figure 2.3).
Figure 2.3: Unsaturated fatty acids tails with kinks.
According to Cerolini et al. (2001), the fresh boar spermatozoa showed two major lipid compositions which are phospholipids and free cholesterol. However, after thawing the total lipid increase indicating that the viability of spermatozoa before freezing is a determinant factor for cellular lipid enrichment from the surrounding medium.
Cholesterol is found in animal derived foods such as eggs (McGuire and Beerman, 2007). According to Lee and Arunasalam (2005), cholesterol is a component of cell membrane and helps to maintain the fluidity of cell membrane. At moderate warm temperatures, the cholesterol molecules reduce the free movement of the phospholipids molecules and make the membrane less fluid. Cholesterol molecules also aid in preventing the close packing of phospholipids molecules and slow down solidification of the membrane. When above transition temperature, phospholipids are able to move certain distances where as below transition temperatures, movement of the molecules is greatly restricted.
There is reduction in the free cholesterol content occurred in boar spermatozoa cell membrane after freezing and thawing processes (Cerolini et al., 2001). The loss in sperm cholesterol content is a feature of capacitation (Therien et al., 1999). Cryopreservation also increases the proportion of capacitated spermatozoa (Watson, 1995). The indicating a loss of cholesterol, is a characteristic event of capacitation, in cryopreserved spermatozoa (Cerolini et al., 2001).
Capacitation is a process that takes place in vivo after the spermatozoa have been present in the female tract for a period of time or in vitro under certain conditions and media. An essential feature of capacitation is the removal of cholesterol from the acrosomal membrane of sperm (Martinez and Morros, 1996). Capacitation has been referred to as changes that enable the sperm to undergo both the acrosome reaction and hyperactivation. Capacitation has been correlated with changes in sperm intracellular ion concentration, plasmic membrane fluidity, metabolism and motility. The free cholesterol:phospholipid ratio in the boar spermatozoa membrane was halved after freezing and thawing because of the decrease in the free cholesterol content (Cerolini et al., 2001).
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The media in which the sperm are incubated play an integral role in many sperm processes (Visconti et al., 1995) such as cryopreservation. According to Cerolini et al. (2001), there are no triacylglycerols detected in fresh spermatozoa. After thawing the boar sperm there are increase in the amount of total lipid. This is due to a major increase in triacylglycerol content. Triacylglycerols are well represented in egg yolk and an interaction between gametes and the egg yolk-based diluents is suggested as the major cause of the described changes in lipid composition. An active cellular lipid metabolism responsible for lipid transfer or synthesis occurs during incubation of spermatozoa within the egg yolk based diluents or after the freezing and thawing procedure.
Lipoproteins are complex globular structure containing varying amounts of triglycerides, phospholipids, cholesterol esters, free cholesterol and protein. The ratio of lipids to proteins determines a lipoprotein’s density and its name. Lipoprotein with relatively more lipid than protein have lower densities (low-density lipoprotein, LDL) compared to those with more protein and less lipid (high-density lipoprotein, HDL) (McGuire and Beerman, 2007).
Egg yolk is a very well known plasma membrane protector and the effect of its low density lipoproteins (LDL) as membrane stabilizers has been widely reported (Hu et al., 2008). LDL contained in egg yolk is largely responsible for sperm protection during cryopreservation (Pace and Graham, 1974).
According to Quinn et al. (1980), LDL adheres to sperm membrane and provides protection to sperm by stabilizing the membrane. Membrane protection is shown by the fact that the increased concentration of LDL results in a linear increase in viability. This protective effect was related to the gelation capacity of LDL microspheres and the inclusion of phospholipids and triglycerides in the sperm membrane can protect the sperm membrane against ice formation by forming a film around it.
Commercial Chicken Egg
Most of the commercial layer chicken farm either reared the chicken in commercial cages or in closed house. Eggs contain with all nine essential amino acids (EAA) (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine), making them an excellent source of high biological value protein (Kassis et al., 2010).
Free-range Chicken Egg
Free-range chickens are reared on pastures or housed in movable pens that are rotated frequently to maximize access to fresh pasture and protects the birds from predators (Long and Alterman, 2007). “Kampung” chicken in Malaysia is also reared in free-range in the villages.
Comparison of Commercial and Free-Range Chicken Egg
The difference in egg nutrient levels in commercial and “kampung” chicken is most likely resulted from the difference in diets consumed by them. Free-range chicken such as “kampung” chicken eat natural diets such as all kinds of seeds, green plants, herbs and insects. On the other hand, the commercial chickens are fed with the possible mixture of corn, soybean mean and all kinds of addictives (Long and Alterman, 2007). The free-range chicken raised outdoors and has daily access to outdoors and physical activities which enables the chicken to find natural diets in the pasture. However, commercial layer chicken production kept the chicken in crowded spaces and completes indoor surrounding throughout the lifetime of the chicken (Long and Alterman, 2007).
Fat, including cholesterol and saturated fat, is contained within the yolk; while egg white contains a negligible amount of fat (Kassis et al., 2010).
These two different rearing systems can result in differing egg weight, yolk weight, fat and cholesterol content. Free range system show lighter in egg weight but more egg yolk content in an egg compared to the commercial rearing system that used cages. Therefore, more egg yolk content in an egg, more fat and cholesterol content in an egg as shown in the free-range rearing system (Simcic et al., 2008).
An inverse relationship between yolk cholesterol concentration and egg weight was observed among avian species with an exception of quail (Choi et al., 2001). This is also show in Isa Brown as the lighter the egg weight, the more yolk cholesterol concentration (Mojca et al., 2008).
According to Long and Alterman, 2007, free-range egg or pasture egg is richer in nutrients than typical supermarket (commercial) eggs. Eggs from hens raised on pasture contain 1/3 less cholesterol, 1/4 less saturated fat, 2/3 more vitamin A, two times more omega-3 fatty acids, three times more vitamin E and seven times more beta carotene compared to the United State Department of Agriculture (USDA) nutrient data for standard commercial eggs.
According to MARDI (2005), Katjang is also known as “Kacang” is one of the local indigenous goat breed that is found in Malaysia. Katjang is also found in Indonesia and Philipinnes. Katjang goat is a prolific breed as twinning is common. Katjang is a non-seasonal goat which enables breeding activities throughout the year. Moreover, Katjang goat is a hardy animal that is capable to survive in different environments conditions. Malaysia Katjang is black or brown in colour. Sometimes white patches are found on the body and leg of the goat. Katjang is meat goat which has a short body shape. The weight of the mature buck is between 25 to 30 kg while the mature doe is between 20 to 25 kg. The carcass yield of Katjang goat is between 44 to 51%.
According to ATTRA (2010), buck is significant as it provides half of the genetics of the herd, which can make significant improvements to the herd. A general physical exam shall be performed in order to check the buck for structural soundness and abnormalities in the sex glands and organs. In general, the scrotal circumference meat bucks shall measure 26 to 29 cm at 45 kg and larger bucks should measure at least 34 to 36 cm. A normal sperm concentration is 2 billion sperm per mililiter of semen with 70% motile and in a straight progressive movement. At least 80% of the sperm are normal. A full-grown, healthy buck should easily service up to 50 does.
The morphology of mature spermatozoa of different species of farm mammals is similar (Campbell et al., 2003). The main components of sperm cell are divided into three parts which are head, midpiece and tail. Nucleus also is located at the head region. The shape of the nucleus is either oval or pear-shaped. The head contained acrosome which have lysosome enzymes to digest the outer membranes of the egg (female gamete) (Lee and Arunasalam, 2005). Acrosome is form when the Golgi complex engenders the vesicles and inverts itself llike a cap over the largest part of the nucleus (Affolter et al., 1999).The head is bound to the midpiece which contained mitochondria which produced energy to the tail for locomotion function.
According to Rouge and Bowen (2002), the most common method use to collect the semen from the buck is artificial vagina (AV). AV had widely use in semen collection for many domestic animals such as cattle, horses, sheep and goats. AV is composed of a tube with an outer rubber lining that hold water at (42 to 48â-‹C) and inner rubber lining that is lubricated.
According to Stephen (2007), the basic design of an AV is the same for all species but minor adaptations may be necessary to reflect the anatomical features of the female reproductive tract. A trained buck shall be trained for semen ejaculation frequently so that the buck gets used to used of AV. Normally a trained buck will readily mount a teaser and as its penis erected, it is directed into the AV. Teaser is a doe which make the AV process must be monitor carefully so no natural mating occurred. Teaser also can be a castrated male.
All the semen collected must be examined to ensure the quality and quantity such as semen volume, pH, colour, total sperm number ejaculate, sperm motility, sperm morphology, sperm viability and sperm membrane integrity. This is because all this parameters are positively correlated with fertility (Panhwar, 2010). The normal values of semen parameters are shown in (Table 2.1).
Computer Assisted Semen Analyzer (CASA)
According to WHO (2010), CASA are capable of measuring sperm motility and kinematics, and some can also be used to estimate sperm concentration. There are few CASA machines, which have semi-automated morphology modules. CASA can use for assessment of motility, concentration and morphology. The major advantages of using CASA over manual methods are high precision on quantitative data on the kinematic parameters of spermatozoa (forward progression and hyperactivated motility, characteristic of capacitated cells).
According to Rrumbullaku (2009), in recent years, a number of techniques for objective assessment of movement characteristics of spermatozoa were introduced by using CASA systems. CASA is a simple classification system which provides the best possible assessment of sperm motility without resorting to complex equipment. The microscopic field is scanned systematically and the motility of each spermatozoon encountered is graded according into 4 types of motility which are rapid progressive motility, slow or sluggish progressive motility, non-progressive motility and immotility.
According to WHO (2010), different CASA instruments use different mathematical algorithms to compute many of these movement variables. The comparability of measurements across all instruments is not yet known.
According to Roy, 1957, fresh semen that need to undergoes the cryopreservation process needed to be centrifuged to remove the seminal plasma. This is because the direct dilution of the fresh semen with the semen extender can cause detrimental effect to the sperm cells. Fresh semen contained seminal plasma which functions as the protector and buffer fluid to the sperm cell while the semen extender contains egg yolk as the cryoprotectant ingredients for the cryopreservation. The mixture of the fresh semen and semen extender shown a harmful interaction between seminal plasma and egg yolk can be detrimental to the sperm cell. This is because the egg yolk has an enzyme known as egg-yolk coagulater enzyme (EYCE). The EYCE is originated from the bulbourethal gland. The EYCE will act as a catalyst that hydrolyzes egg yolk lecithin into fatty acids and lysolecithin (Iritani and Nishikawa, 1961).
Sperm membrane is more fusogenic due to the hydrolysis process. Thus, this will trigger the acrosome reaction (Upreti et al., 1999) and chromatin de-condensation (Sawyer and Brown, 1995). Hence, sperm cells maintained their motility in egg yolk diluents if the seminal plasma was removed (Roy, 1957).
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