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Penetrating Cryoprotectants Across Cell Membranes Biology

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Chapter 2


Cryopreservation of gametes is of import because it would let us to back up a genome resource bank for a strain for an indefinite period of clip. Cryopreservation as a technique of storage of caprine animal sperm has advantages but stop deading and dissolving induces damaging effects in footings of sperm ultrastructural, biochemical and functional harm, [ 1 ] P.F. Watson, The causes of decreased birthrate with cryopreserved seeds, Anim Reprod Sci 60-61 ( 2000 ) , pp. 481-492. Article | PDF ( 155 K ) | View Record in Scopus | Cited By in Scopus ( 227 ) ensuing in a decrease of motility, membrane unity and fertilizing ability ( Purdy, 2006 ) .

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A cryoprotectant is included in a cryopreservation medium to minimise the physical and chemical emphasiss ensuing from the chilling, freeze, and melt of sperm cells ( Purdy, 2006 ) . Cryoprotectants are usually categorized into two wide categories based on their ability to spread across cell membranes, which are perforating cryprotectant or non-penetrating cryoprotectant ( Muldrew, 1998 ) . The colligative action of both perforating and non-penetrating cryoprotectant allows the cell to last the status for a decrease of cell H2O content during chilling thereby cut downing the sum of intracellular freeze ( Mc Gann, 1978 ) .

This can be shown in the combination of glycerin, dimethyl sulfoxide ( DMSO ) and dextran ( Kundu et al. , 2002 ) .

Penetrating Cryoprotectants

Penetrating cryoprotectants are able to travel across cell membranes ( Muldrew, 1998 ) and make the environment for a decrease of cell H2O content at temperatures sufficiently low to cut down the detrimental consequence of the concentrated solutes on the cells ( Mc Gann, 1978 ) . This is because the penetrating cryoprotectants force the H2O to be drawn out osmotically from the cell such as sperm cell. As the sperm cell now has less intracellular H2O, less intracellular ice formation will happen when the stop deading point of

the cell is decreased. This is good as intracellular ice can ensue in cell decease and accordingly reduced birthrate of the seeds. The penetrating cryoprotectant besides cause membrane lipoid and protein rearrangement, which consequences in increased membrane fluidness, greater desiccation at lower temperatures and hence an increased ability to last for cryopreservation procedures ( Holt, 2000 ) . Penetrating cryoprotectants are dissolvers that dissolve sugars and salts in the cryopreservation medium ( Purdy, 2006 ) .

There are many perforating cryoprotectants such as glycerin, dimethyl sulfoxide ( DMSO ) , ethylene ethanediol, propene ethanediol ( Leboeuf et al. , 2000 ) and dimethyformamide ( Meryman, 1971 ) .

Glycerol was the first Cryoprotectant Agent ( CPA ) that gained widespread usage in cryobiology for ruddy blood cell and sperm cell ( Best, 2008 ) and is the most normally used perforating cryoprotectant ( Purdy, 2006 ) . However, glycerin has a failing with its slow motion across those membranes which are permeable to it ( Meryman, 1971 ) . Therefore, big figure of tissues or cells will look to be virtually impermeable. By and large approximately 1 to 8 per centum glycerin is used and it showed a great recovery of sperm post-thawing ( Singh et al. , 1995 ) .

On the other manus, DMSO shows good properties in the incursion velocity. Ehtylene glycol maps as an car anti-freeze while propene ethanediol was at first used to cut down formation of ice in ice pick besides shows to be an effectual cryoprotectant ( Best, 2008 ) . Formamide need to be assisted by other cryoprotectants in order to vitrify. High concentration of perforating cryoprotectants usage for the intent of cells protection such as DMSO, ethene ethanediol and dimethyformamide can do toxicity ( Meryman, 1971 ) . The more in the figure of methyl group ( R-CH3 ) , the stronger is the cryoprotectants ( Best, 2008 ) .

2.2.2 Non-penetrating Cryoprotectants

Non-penetrating cryoprotectants can non travel across cell membranes ( Muldrew, 1998 ) , therefore it osmotically “ squeezing ” H2O from the cells chiefly during the initial stages of stop deading at temperatures between a?’10 and a?’20 A°C when these cryoportectant become concentrated in the extracellular parts ( Mc Gann, 1978 ) . The high sub-freezing temperatures will do the non-penetration cryoprotectants desiccate the cell and let a rapid chilling alternatively of slow chilling. This is because slow chilling can take to extended harm on the cell ( Muldrew, 1998 ) . Good non-penetrating cryoprotectants besides shall hold the reversible ability in osmolarity although under osmotic emphasis ( Meryman, 1971 ) . Non-penetrating cryoprotectants are by and large comprised from polymers that can organize extended H bonds with H2O ( Muldrew, 1998 ) .

Egg yolk is a common constituent of most seeds cryopreservation extenders for domestic animate beings ( Tuli and Holtz, 1994 ) . Variation from 5 to 50 per centum sum of egg yolk used appear to give good consequences but 20 per centum or less are more common concentration of egg yolk used today ( Campbell et al. , 2003 ) . It has been shown to hold a good consequence on sperm cryopreservation. This is because egg yolk serves as a protectant of the plasma membrane and acrosome against temperature-related hurt as egg yolk and glycerol-free medium used as cryoprotectant showed a positive consequence of motility after post-thawing the caprine animal sperms ( Kundu et al. , 2001 ) . Egg yolk was found to be the chief cryoprotective agent, but at that place was a interactive consequence between glycerin ( perforating cryoprotectant ) and egg yolk ( non-penetrating cryoprotectant ) in supplying the greatest post-thaw endurance ( 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 besides increased and reached an optimal value. Any farther additions in dextran concentration will ensue and adverse consequence the recovery of motility ( Kundu et al. , 2002 ) .

Artificial Insemination

Artificial insemination ( AI ) involves taking seeds from a male and presenting it into the generative piece of land of a female, by agencies of a catheter, at a suited phase in her generative rhythm ( Stephen, 2007 ) . AI was the first biotechnology applied to better reproduction and genetic sciences of farm animate beings ( Amer et al. , 2008 ) .

Harmonizing to Leboeuf et Al. ( 2000 ) , AI has an of import function in caprine animal genteelness, particularly in intensive systems of production, to command reproduction and, in concurrence with accurate offspring testing, to better the production of milk, hair and meat. AI includes more efficient familial choice strategies and the use and storage of the familial stuff.

The seeds collected will be subjected to seeds rating before insemination procedure is taken. Department of energies can be inseminated with either fresh seeds or commercially available frozen seeds ( Allison and Hagevort, 2009 ) . Artificial insemination remains the chief vehicle for the rapid airing of valuable cistrons and the method of pick for the dairy husbandman worldwide to better the familial quality of their farm animal ( Vishwanath and Shannon, 2000 ) . The success of an AI plan depends on the proper direction of seeds aggregation, storage and usage ( Leboeuf et al. , 2000 ) .

Chicken Egg

Egg is one of the best and most cheap beginnings of high quality protein and contains a balanced distribution of assorted minerals and vitamins. An egg contains yolk, vitalline membrane, chalazae, albumins, air cell, inner and outer membranes and shell ( Figure 2.1 ) . The cholesterin content of eggs is influenced by familial factors, dietetic composing, lay strength, layer age and medical intervention ( Vorlova et al. , 2001 ) .

Figure 2.1: Cross-section of a freshly laid egg.

Egg Yolk

Egg yolk is one of the common constituents of most seeds cryopreservation extenders for domestic animate beings. Chicken egg yolk systematically improved the endurance and the care of the fertilizing ability of sperm cell when included in the preservation medium ( Trimeche et al. , 1997 ) . The fatty substances of the yolk are largely triglycerides ( true fat ) 65.5 % , phospholipids 28.3 % , and cholesterol 5.2 % ( USDA, 2000 ) . Egg yolk contains phospholipids, cholesterin and low denseness lipoproteins which are the factors that provide protection to sperm against cold daze during the freeze-thaw procedure ( KulaksA±z et al. , 2010 ) . The best quality gametes after cryopreservation are enriched in lipid, triacylglycerol and phospholipid ( Cerolini et al. , 2001 ) .


Phospholipids contain a glycerin molecule bonded to two fatty acids and a phosphate-containing polar caput group ( Figure 2.2 ) . Phospholipids are amphipathic as it contains both polar ( hydrophilic ) and nonionic ( hydrophobic ) parts. The hydrophobic part consists of two fatty acids whereas the hydrophilic part is the polar caput group. This construction allows phospholipids to be a major constituent of cell membranes ( McGuire and Beerman, 2007 ) . Saturated fatty acids have no dual bonds between C atoms and illustrations of concentrated fat are palmitic acid and stearic acid. Unsaturate fatty acids have one or more dual bonds between C atoms ( -C=C- ) which cause cricks in the concatenation 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 caput indicating to the extra- and intracellular infinites that provide stable barrier for the cell ( McGuire and Beerman, 2007 ) . Harmonizing to Lee and Arunasalam ( 2005 ) , cell membrane is dynamic, unstable constructions. The fluidness of the cell membranes can be influenced by the motion of phospholipids, types of fatty acids in the membranes, cholesterin and temperature. Phospholipids molecules can travel laterally or alter topographic points ( reversal ) and therefore give the membrane fluidness. Lateral motion happened quickly where as flip-flopping across the membrane occurs at a really slow rate.

Harmonizing to Mazur ( 1984 ) , membranes are destabilized by several factors which are the transition to and from the storage temperature ( thermic emphasis ) , the big volume alterations associated with H2O and cryoprotectant motion and the exposure to high salt concentrations ( osmotic emphasis ) . At a molecular degree, alterations in membrane organisation such as alterations of specific lipid-protein interaction, phospholipids dissymmetry and lipid composing are the deduction in the loss of cell membrane permeableness such as sperm cell. The reversibility of these alterations could be dependent on membrane kineticss and the physical belongingss of the membranes have been assumed to be one of the polar factors in the opposition of sperm cell to cryopreservation ( Hammerstedt et al. , 1990 ) . Sperm plasma membrane is a cardinal cell organ in commanding sperm cryosurvival ( Giraud et al. , 2000 ) .

Harmonizing to Cerolini et Al. ( 2001 ) , polyunsaturated fatty acids in frozen-thawed Sus scrofa sperm cell decreased because comparative addition in the proportions of concentrated owing to the emphasis of cryopreservation as sperm cell membrane may damage. This is because unsaturated fatty acids have dual bonds which form cricks to forestall close wadding of the phospholipids molecules, addition fluidness and promote motion of substances across the membrane ( Lee and Arunasalam, 2005 ) ( Figure 2.3 ) .

Figure 2.3: Unsaturated fatty acids dress suits with cricks.

Harmonizing to Cerolini et Al. ( 2001 ) , the fresh Sus scrofa sperm cell showed two major lipid composings which are phospholipids and free cholesterin. However, after dissolving the entire lipid addition indicating that the viability of sperm cell before freeze is a deciding factor for cellular lipid enrichment from the environing medium.


Cholesterol is found in animate being derived nutrients such as eggs ( McGuire and Beerman, 2007 ) . Harmonizing to Lee and Arunasalam ( 2005 ) , cholesterin is a constituent of cell membrane and helps to keep the fluidness of cell membrane. At moderate warm temperatures, the cholesterin molecules cut down the free motion of the phospholipids molecules and do the membrane less fluid. Cholesterol molecules besides aid in forestalling the close wadding of phospholipids molecules and decelerate down hardening of the membrane. When above passage temperature, phospholipids are able to travel certain distances where as below passage temperatures, motion of the molecules is greatly restricted.

There is decrease in the free cholesterin content occurred in Sus scrofa sperm cell cell membrane after stop deading and dissolving procedures ( Cerolini et al. , 2001 ) . The loss in sperm cholesterin content is a characteristic of capacitation ( Therien et al. , 1999 ) . Cryopreservation besides increases the proportion of capacitated sperm cell ( Watson, 1995 ) . The bespeaking a loss of cholesterin, is a characteristic event of capacitation, in cryopreserved sperm cell ( Cerolini et al. , 2001 ) .

Capacitation is a procedure that takes topographic point in vivo after the sperm cell have been present in the female piece of land for a period of clip or in vitro under certain conditions and media. An indispensable characteristic of capacitation is the remotion of cholesterin from the acrosomal membrane of sperm ( Martinez and Morros, 1996 ) . Capacitation has been referred to as alterations that enable the sperm to undergo both the acrosome reaction and hyperactivation. Capacitation has been correlated with alterations in sperm intracellular ion concentration, plasmic membrane fluidness, metamorphosis and motility. The free cholesterin: phospholipid ratio in the Sus scrofa sperm cell membrane was halved after stop deading and dissolving because of the lessening in the free cholesterin content ( Cerolini et al. , 2001 ) .

The media in which the sperm are incubated play an built-in function in many sperm procedures ( Visconti et al. , 1995 ) such as cryopreservation. Harmonizing to Cerolini et Al. ( 2001 ) , there are no triacylglycerols detected in fresh sperm cell. After dissolving the Sus scrofa sperm there are addition in the sum of entire lipid. This is due to a major addition in triacylglycerol content. Triacylglycerols are good represented in egg yolk and an interaction between gametes and the egg yolk-based dilutants is suggested as the major cause of the described alterations in lipid composing. An active cellular lipid metamorphosis responsible for lipid transportation or synthesis occurs during incubation of sperm cell within the egg yolk based dilutants or after the freeze and thawing process.


Lipoproteins are complex ball-shaped construction incorporating changing sums of triglycerides, phospholipids, cholesterin esters, free cholesterin and protein. The ratio of lipoids to proteins find a lipoprotein ‘s denseness and its name. Lipoprotein with comparatively more lipid than protein have lower densenesss ( 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 really good known plasma membrane defender and the consequence of its low denseness lipoproteins ( LDL ) as membrane stabilizers has been widely reported ( Hu et al. , 2008 ) . LDL contained in egg yolk is mostly responsible for sperm protection during cryopreservation ( Pace and Graham, 1974 ) .

Harmonizing to Quinn et Al. ( 1980 ) , LDL adheres to sperm membrane and provides protection to sperm by stabilising the membrane. Membrane protection is shown by the fact that the increased concentration of LDL consequences in a additive addition in viability. This protective consequence 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 organizing a movie around it.

Commercial Chicken Egg

Most of the commercial bed chicken farm either reared the poulet in commercial coops or in closed house. Eggs contain with all nine indispensable amino acids ( EAA ) ( histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine ) , doing them an first-class beginning of high biological value protein ( Kassis et al. , 2010 ) .

Free-range Chicken Egg

Free-range poulets are reared on grazing lands or housed in movable pens that are rotated often to maximise entree to fresh grazing land and protects the birds from marauders ( Long and Alterman, 2007 ) . “ Kampung ” poulet in Malaysia is besides reared in free-range in the small towns.

Comparison of Commercial and Free-Range Chicken Egg

The difference in egg food degrees in commercial and “ kampung ” chicken is most likely resulted from the difference in diets consumed by them. Free-range poulet such as “ kampung ” chicken eat natural diets such as all sorts of seeds, green workss, herbs and insects. On the other manus, the commercial poulets are fed with the possible mixture of maize, soya bean mean and all sorts of addictives ( Long and Alterman, 2007 ) . The free-range poulet raised out-of-doorss and has day-to-day entree to out-of-doorss and physical activities which enables the poulet to happen natural diets in the grazing land. However, commercial bed chicken production kept the poulet in crowded infinites and completes indoor environing throughout the life-time of the poulet ( Long and Alterman, 2007 ) .

Fat, including cholesterin and saturated fat, is contained within the yolk ; while egg white contains a negligible sum of fat ( Kassis et al. , 2010 ) .

These two different rise uping systems can ensue in differing egg weight, yolk weight, fat and cholesterin content. Free scope system show igniter in egg weight but more egg yolk content in an egg compared to the commercial raising system that used coops. Therefore, more egg yolk content in an egg, more fat and cholesterin content in an egg as shown in the free-range raising system ( Simcic et al. , 2008 ) .

An opposite relationship between yolk cholesterin concentration and egg weight was observed among avian species with an exclusion of quail ( Choi et al. , 2001 ) . This is besides show in Isa Brown as the igniter the egg weight, the more yolk cholesterin concentration ( Mojca et al. , 2008 ) .

Harmonizing to Long and Alterman, 2007, free-range egg or grazing land egg is richer in foods than typical supermarket ( commercial ) eggs. Eggs from biddies raised on grazing land contain 1/3 less cholesterin, 1/4 less concentrated fat, 2/3 more vitamin A, two times more omega-3 fatty acids, three times more vitamin E and seven times more beta provitamin A compared to the United State Department of Agriculture ( USDA ) food informations for standard commercial eggs.

Katjang Goat

Harmonizing to MARDI ( 2005 ) , Katjang is besides known as “ Kacang ” is one of the local autochthonal caprine animal strain that is found in Malaysia. Katjang is besides found in Indonesia and Philipinnes. Katjang caprine animal is a fecund strain as twinning is common. Katjang is a non-seasonal caprine animal which enables genteelness activities throughout the twelvemonth. Furthermore, Katjang caprine animal is a stalwart animate being that is capable to last in different environments conditions. Malaysia Katjang is black or brown in coloring material. Sometimes white spots are found on the organic structure and leg of the caprine animal. Katjang is meat caprine animal which has a short organic structure form. The weight of the mature vaulting horse is between 25 to 30 kilograms while the mature Department of Energy is between 20 to 25 kilogram. The carcass output of Katjang caprine animal is between 44 to 51 % .

Harmonizing to ATTRA ( 2010 ) , buck is important as it provides half of the genetic sciences of the herd, which can do important betterments to the herd. A general physical test shall be performed in order to look into the vaulting horse for structural soundness and abnormalcies in the sex secretory organs and variety meats. In general, the scrotal perimeter meat vaulting horses shall mensurate 26 to 29 centimeter at 45 kilograms and larger vaulting horses should mensurate at least 34 to 36 centimeter. A normal sperm concentration is 2 billion sperm per mililiter of seeds with 70 % motile and in a consecutive progressive motion. At least 80 % of the sperm are normal. A adult, healthy vaulting horse should easy serve up to 50 does.

Sperm Morphology

The morphology of mature sperm cell of different species of farm mammals is similar ( Campbell et al. , 2003 ) . The chief constituents of sperm cell are divided into three parts which are caput, midpiece and tail. Nucleus besides is located at the caput part. The form of the karyon is either egg-shaped or pear-shaped. The caput 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 cysts and inverts itself llike a cap over the largest portion of the karyon ( Affolter et al. , 1999 ) .The caput is bound to the midpiece which contained chondriosomes which produced energy to the tail for motive power map.

Semens Collection

Harmonizing to Rouge and Bowen ( 2002 ) , the most common method usage to roll up the seeds from the vaulting horse is unreal vagina ( AV ) . AV had widely use in seeds aggregation for many domestic animate beings such as cowss, Equus caballuss, sheep and caprine animals. AV is composed of a tubing with an outer gum elastic liner that hold H2O at ( 42 to 48a-‹C ) and interior gum elastic liner that is lubricated.

Harmonizing to Stephen ( 2007 ) , the basic design of an AV is the same for all species but minor versions may be necessary to reflect the anatomical characteristics of the female generative piece of land. A trained vaulting horse shall be trained for seeds ejaculation often so that the vaulting horse gets used to used of AV. Normally a trained vaulting horse will readily mount a teaser and as its phallus erected, it is directed into the AV. Teaser is a Department of Energy which make the AV procedure must be monitor carefully so no natural coupling occurred. Teaser besides can be a unsexed male.

Semens Evaluation

All the seeds collected must be examined to guarantee the quality and measure such as seeds volume, pH, coloring material, entire sperm figure semen, sperm motility, sperm morphology, sperm viability and sperm membrane unity. This is because all this parametric quantities are positively correlated with birthrate ( Panhwar, 2010 ) . The normal values of seeds parametric quantities are shown in ( Table 2.1 ) .

Table 2.1: Normal values of fresh human seeds variables

Semens variables

Standard Values


2.0 milliliter or more



Sperm Concentration

20 ten 106 spermatozoa/ml or more

Entire Sperm Count

40 ten 106 spermatozoa per semen or more


50 % or more with forward patterned advance ( rapid progressive motility or decelerate or sluggish progressive motility ) or more with rapid imperfect within 60 proceedingss of interjection


30 % or more with normal signifiers


75 % or more unrecorded sperm cell

Beginning: WHO, 1992

Computer Assisted Semen Analyzer ( CASA )

Harmonizing to WHO ( 2010 ) , CASA are capable of mensurating sperm motility and kinematics, and some can besides be used to gauge sperm concentration. There are few CASA machines, which have semi-automated morphology faculties. CASA can utilize for appraisal of motility, concentration and morphology. The major advantages of utilizing CASA over manual methods are high preciseness on quantitative informations on the kinematic parametric quantities of sperm cell ( forward patterned advance and hyperactivated motility, feature of capacitated cells ) .

Sperm Motility

Harmonizing to Rrumbullaku ( 2009 ) , in recent old ages, a figure of techniques for nonsubjective appraisal of motion features of sperm cell were introduced by utilizing CASA systems. CASA is a simple categorization system which provides the best possible appraisal of sperm motility without fall backing to complex equipment. The microscopic field is scanned consistently and the motility of each sperm cell encountered is graded harmonizing into 4 types of motility which are rapid progressive motility, decelerate or sluggish progressive motility, non-progressive motility and immotility.

Harmonizing to WHO ( 2010 ) , different CASA instruments use different mathematical algorithms to calculate many of these motion variables. The comparison of measurings across all instruments is non yet known.


Harmonizing to Roy, 1957, fresh seeds that need to undergoes the cryopreservation procedure needed to be centrifuged to take the seminal plasma. This is because the direct dilution of the fresh seeds with the seeds extender can do damaging consequence to the sperm cells. Fresh seeds contained seminal plasma which maps as the defender and buffer fluid to the sperm cell while the seeds extender contains egg yolk as the cryoprotectant ingredients for the cryopreservation. The mixture of the fresh seeds and seeds extender shown a harmful interaction between seminal plasma and egg yolk can be damaging 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 secretory organ. The EYCE will move as a accelerator that hydrolyzes egg yolk lecithin into fatty acids and lysolecithin ( Iritani and Nishikawa, 1961 ) .

Sperm membrane is more fusogenic due to the hydrolysis procedure. Therefore, this will trip the acrosome reaction ( Upreti et al. , 1999 ) and chromatin de-condensation ( Sawyer and Brown, 1995 ) . Hence, sperm cells maintained their motility in egg yolk dilutants if the seminal plasma was removed ( Roy, 1957 ) .

Cite this Penetrating Cryoprotectants Across Cell Membranes Biology

Penetrating Cryoprotectants Across Cell Membranes Biology. (2017, Jul 09). Retrieved from https://graduateway.com/penetrating-cryoprotectants-across-cell-membranes-biology-essay/

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