The procedure of cryopreservation has been universally accepted for continuing the biological stuffs for eternity old ages ( Chen, 1988 ) . In theory, the viability of the cryopreserved samples could be retained with no structural alterations. Bing the natural component H2O plays an indispensable function by bearing the biochemical activities within the cells. Ever since, H2O has been proved to hold a enormous function in debasement of stored stuffs ( Adams, 2007 ) the surveies on transition of H2O in to frost and its deadly effects has besides received adequate attending.
The rule of cryopreservation is to use extremist low temperature to suspend the biochemical reaction of the samples. By and large, cryopreservation employs utilizing liquid N temperature ( -196C ) and at this temperature no biochemical reaction takes topographic point. However, it is non possible to cut down the temperature successfully to such low temperature because H2O inside the tissues is forced to organize ice when its reaches its freezing point. Although, the stop deading point of H2O is non precisely 0C, and the procedure of ace chilling tend to be an of import phenomenon in most of the samples lasting low temperature. Super chilling is the procedure by which the cellular H2O is seized from stop deading and when the temperature lowers any farther may ensue in forced ice formation.
In cryobiology, the great challenge is to take down the temperature to glaze passage temperature ( Tg ) because most of the samples have no activity when the H2O inside the samples are later converted to glaze. Glass is an formless solid, and at that place has been arguments refering the smaller activity of the samples that could take to sample debasement over a period of clip. In wider context, most of the samples posses glass passage around temperatures -130C and to be really precise any temperature below the glass passage temperature could give longer viability than the samples held at liquid N vapour stage temperature i.e. -130C. This is the ground for taking liquid N temperature whilst set uping longer cryobiological protocols.
Clearly, non all samples amend to the cryo-banking and for cut downing the temperature to the temperature of involvement ; cryoprotectants go an indispensable function ( Watson et al. , 1985 ) . It has been extremely recognized that the simple freeze and melt of the cells leads to cell harm and this is believed to happen because of the ice nucleation and desiccation ( Mazur, 1970 ) . The aim of utilizing the cryoprotectant during freeze is to keep the H2O with out organizing ice until the glass passage temperature is reached. Although, such additives can non ensue in 100 % endurance after stop deading and dissolving, but in bulk of the instances the freezing hurt tend to be suppressed ( Fahy, 1985 ) . The cryoprotectants used must sufficiently concentrate to supercool and solidify into a glass and the H2O must non be converted to frost readily and alternatively it would be converted to glaze by the procedure of vitrification ( Rall, 1985 ) . There are two different type of cryoprotectants being used for cryopreservation technique. The permeating cryoprotectant normally used for sperm cryopreservation could perchance pervade in to the cells and bring forth important consequence. The most normally used perforating cryoprotectant includes glycerin and dimethyl sulfoxide ( DMSO ) is the best illustration ( Li et al. , 2005 ) . The other type of cryoprotectant has the inclination non to come in inside the membranes and it remains in the extracellular infinite ( Sztein et al. , 2001 ) .
In most of the cryopreservation techniques the permeating cryoprotectant are permeated in to the cells. The entry of the cryoprotectant is believed to go on by thermodynamic equilibration and it besides enhances the remotion of pervading cryoprotectants during dissolving ( Elmoazzen et al. , 2005 ) . Ethylene glycol finds its of import application in cryopreserving mammalian embryo freeze because it carries low expression weight and high pervasion in to cells when compared with other cryoprotectant ( Chi et al. , 2002 ) .
Urgent demands for both works and animate being preservation have attracted batch of the preservation scientists to concentrate on cryopreservation. Indeed, the cryopreservation is a dependable method for preservation and requires less money and less clip consuming. The field expanded in conserving the endangered fish species through out the universe, and the aquatic species figure has been worsening bit by bit. The sperm cryopreservation of zebra fish has been proved to be possible guaranting the species preservation, the cryopreservation of oocyte and embryo still remains a greater challenge ( Guan et al. , 2008 ) . Recently the zebra fish and other fish tank fish have suit to be the most critical theoretical account system species for the surveies ( Robles et al. , 2009 ) . In zebra fish creative activity of cryobanks of feasible fish sperm, oocytes and embryos are of of import demand. Although the storage clip of cryobanks is unpredictable the liquid N storage of fish cells could last between 200 and 32,000 old ages ( Robles et al. , 2009 ) .
Despite of successful cryopreservation of sperm cell of many species of fish ( e.g. cyprinids, salmonid etc. ) are in pattern, the systemic surveies on fish oocytes cryopreservation is still missing ( Chen and Tian, 2005 ) . However, few embryos have been concluded to with stand short period of decrease in temperature to liquid nitrogen temperature and the consistence in bring forthing the same consequences remains undefinable ( Isayeva et al. , 2004 ) . The maternal genome cryopreservation of the worlds has been shown to be possible ; the successful endurance of maternal genome of fish still remains disputing. Furthermore, maternal genome cryopreservation seems to be really important in aquatic species because there are several familial factors that are being transformed motherly through oocyte cytol including mitochondrial Deoxyribonucleic acid and mRNS & A ; acirc ; ˆ™s that plays a critical function in early embryonic development ( Zhang et al. , 2008 ) . Continuing such cardinal cistrons can besides better the opportunity of conserving the species where the oocyte and embryo cryopreservation fails like in zebrafish.
The handiness of eggs that are located in the cerebral mantle with in the aboriginal follicles, can be isolated easy because it is really near to the surface ( Lass et al. , 1996 ) . The oarian cerebral mantle encloses most of the aboriginal follicles and it characterizes the resting reserve of germ cells. However, non all the follicles undergo ripening and ovulation, most of the cells die through a procedure known as atresia. Folliclular atresia is a degenerative process through which the ovarian follicles lose their unity and this procedure takes before the ovulation. Furthermore, the ovary of Danio rerio is asynchoronous in nature and the development takes topographic point in four different phases, and it includes primary oocyte growing, cortical air sac phase, vitellogenesis and ripening ( Ucuncu and Cakici, 2009 ) . In fact, more consideration has been given to cryopreserving follicular cells with utilizing those cells for both in vitro and/or in vivo vivo ripening. Since the morphological surveies after stop deading and dissolving are unequal and it is non necessary that the morphology correlates to the follicular development.
The jobs associated with embryo cryopreservation of zebrafish are by and large high yolk content and the scarey hurt ( Isayeva et al. , 2004 ) .The hurt could happen either during freeze or melt. Obviously, the intracellular ice crystal formation breaks the cell organs and cells, Before considereing a cryopreservation technique the equilibrium rate along with freeze and melt rates are to be considered really crucially. However, the oocytes are smaller in size when compared to the eggs and it constitute individual subdivision and have a reduced hazard because of the absence of yold syncytical bed that are seen merely in embryos ( Prescott, 1995 ) . Besides, the fish oocytes possess greater cryoprotectant tolerance bound when compared to embryos ( Plachinta et al. , 2004 ) and the permeableness of the cryoprotectant to the oocytes seems to be more in oocytes and the embryos frequently show lesser pervasion ( ) . The temperature of ice crystal formation and the strength of the cryoprotectant pervasion and the capacity to forestall crystal formation must be critically reviewed before utilizing the cryoprotectant. Furthermore, the cryoprotectant toxicity and the extent to forestall the ice crystal formation may change for each cells and tissue types.
There are two methods noted so far in cryobiology techniques for cut downing the temperature from room temperature to liquid nitrogen temperature. The oldest method that still finds a competitory function in cryopreservation is the controlled slow chilling. Such method utilizations slow chilling rate of about 1 or 2C/hr until the glass passage temperature is reached and this measure is followed is followed by immersing the samples in to liquid N. This method may possibly necessitate cryoprotectant and many protocols defined old has used less cryoprotectant ( Zhang and Rawson, 1995 ) . This is because the slow freeze desiccates the intracellular H2O and therefore accent higher endurance. On the other manus, the other method that has wider application and late accepted is the vitrification. In contrast to command slow chilling the vitrification protocols employ utilizing faster chilling rate and this requires utilizing more concentration of cryoprotectant. The chilling rate by and large performed for vitrification scopes from 40C/hr to 700C/hr ( Kasai et al. , 1990 ; Fahy, 1985 ) . Since the vitrification uses rapid chilling rates and this consequences in hardening of the solution therefore the samples escape from organizing ice-crystals and subsequent cellular harm. However, utilizing utmost concentration of cryoprotectant may be peculiarly toxic to one cell embryos and oocytes ( Mukaida et al. , 1998 ) . Although, both the method finds equal importance in cryopreservation, in rigorous sense, slow chilling rates result in higher endurance for fish embryos than high chilling rates with successful rates runing from 0.01 to 0.75C/min ( Harvey and Ashwood-Smith, 1982 ) .
Furthermore, in matching to the success rates of cryopreservation of specific variety meats of endangered species, the jobs associated with cryodamage has known to do broad harm to the cellular membrane. In add-on, the metabolic activity of the cells seems to be reduced and disrupt the mitochondrial bioenergetical procedures with in the cells ( Kopeika, 2004 ) . Although recent reappraisals clearly project the image of generative cells and its cryodamage in footings of structural changes, the biochemical changes of the cryopreserved generative cells still remains unknown ( Tatone et al. , 2010 ) . The cryopreservation of gamete and embryo has developed greatly by utilizing glycerin as a cryoprotectant, the cognition in heightening the morphological and fuctional endurance has been more. In contrast, the information on the molecular degree alterations of the cryopreserving the oocytes and embryos are still missing.
Most of the cells undergoing cryopreservation demand to be thawed one time required. Most of the cells experience biochemical harm including oxidative emphasis by reactive O species formed upon dissolving ( Park, 1998 ) . Oxidative emphasis seems to be a confining factor in obtaining successful cryopreservation protocol, however, heat daze intervention enhances the cells resistant to damage that occurs from stop deading and dissolving ( Komatsu, 1990 ) . Heat daze proteins may salvage damaged cells. GSH1, GSH2, and trehalose seem to hold an of import function in eliminating oxidative emphasis.
The production and accretion of decreased O intermediates including superoxide groups, vest O, H peroxide and hydroxyl group are frequently grouped under oxidative emphasis. Such emphasis can impact the physiological province of the cryopreserved cells including lipid amendss, protein and DNA amendss ( Lesser, 2006 ) . The formation of oxidative emphasis has become more common in universe & A ; acirc ; ˆ™s oceans and this emphasis has been witnessed to be a consequence of environmental change covering thermic emphasis, UV radiation exposure and pollution effects. In add-on to natural emphasiss, the cryopreservation besides tends to increase the oxidative emphasis. In Saccharomyces cerevisiae ( Yeast ) , Ty1 is a retrotransposon and the heterotaxy at new locations in the host geneome can be expeditiously activated by exposure to UV visible radiation, X-ray, and nitrogen famishment. However recent study by Stamenova et Al. ( 2008 ) showed that controlled measure wise chilling of the cells besides resulted in increased Ty1 degrees. The cells were cooled for 2 hours at 4C and this measure was followed by stop deading for 1 hr at -10C and subsequent chilling by 16 hours at -20C. Clearly, the freeze-induced Ty1 heterotaxy did non took topographic point in mitochondrial mutations and this gives good grounds that the Ty1 heterotaxy resulted on chilling depends greatly on mitochondrial oxidative phosphorylation. However, the increased degree of reactive O species possesses a important function in triping the Ty1 retrotransposon heterotaxy in the frozen barm cells. Reactive O species has been given great concerns and it accumulates while executing the cryopreservation techniques.
The initiation of oxidative emphasis by a pesticide called atrazine ( ATZ ) has been reproted in the liver and ovary samples of the female zebrafish ( Jin et al. , 2010 ) . The GSH and MDA content of the liver was besides found to be altered. The antioxidant enzyme activity besides seems to be reduced. Fish carry similar defence against toxic chemicals like mammals, and to wider context this covers the oxidative emphasis coevals, the written text of certain protective cistrons has been related to be induced by specific DNA motives frequently reffered to as electrophile response elements ( EPREs ) ( Carvan et al.,2001 ) .
However, blastomeres cryopreserved utilizing phosphate buffered saline and in another effort made by equilibration of blastomeres with 2M dimethyl sulfoxide ( Me2SO ) for 1 hr and the other method involved utilizing dimethyl sulfoxide as a cryoprotectant resulted in some mitochondrial DNA mutant ( Kopeika et al. , 2005 ) . The Polymerase Chain Reaction ( PCR ) often used in the cistron mutant analysis has become a revolutionised method and it was used to anlayse the mitochondrial DNA. The venue of the meitnerium DNA were amplified and the comparing between the criterion and experimental consequences revealed the addition in mutant for phosphate buffered saline. However, no important mutant has been increased in the consequence of utilizing 2 M Me2SO4.
Significant consequence of increased per centum of DNA atomization has been noted during the cryopreservation of sperm. Oxidative emphasis biomarker has been used to place the impacts, 8-oxo-7, 8-dihydro-2 & A ; acirc ; ˆ™deoxyguanosine ( 8OHdG ) is the most common biomarker. Apoptotis index would give the per centum caspase of positive cells ( Zribi et al. , 2008 ) . Harmonizing to Perez-Cerezales et Al. ( 2008 ) the harm in the Deoxyribonucleic acid which has been frequently described as a effect of frozen storage may be consequence from oxidative emphasis. The production of DNA harm by oxidative emphasis during sperm cryopreservation has been concluded as a consequence of free extremist coevals. Furthermore, the oxidization emphasis can impact the strands of the Deoxyribonucleic acid and the N bases may acquire altered.
Apoptosis is an of import phenomenon in higher being and it is cardinal biological procedure necessary for normal development and tissue care ( Kerr et al. , 1995 ) . Significant relation between oxidative emphasis changing the go-betweens of cellular procedures including programmed cell death and mortification in Marine being has been noted ( Lesser, 2006 ) . Cooling emphasis as an trigger to cell stress response and later triping the procedure the programmed cell death which leads to mortification is besides clearly understood ( Sonna et al. , 2002 ) . Furthermore, the response of programmed cell death as a consequence of low temperature seems to hold small influence on both the temperature exposed and the continuance of the exposing clip. The chilling emphasis besides consequences in impacting some of the cistrons which besides includes apoptotic specific protein cistron ( ASP ) . In order to analyze the consequence of oxidative emphasis in cryopreservation, mensurating the peculiar cistrons will go more antiphonal in footings of molecular degree survey. In add-on the cold emphasis besides seems to increase E choice ( Cell adhesion cistron ) , HSP cistron, p53 ( Cell-cycle cistron ) and IL-8 ( Cytokine cistron ) ( Boonkuson et al. , 2006 ) .
Methanol will be used as a cryoprotectant for this experiment because early studies suggested the toxicicity of utilizing DMSO in mouse oocytes ( Vincent et al. , 1990 ) . Since there has been no study proposing the induction of oxidative emphasis as a confining factor for oocyte cryopreservation in zebrafish, the survey is intended to analyze the consequence of oxidative emphasis during cryopreservation of zebrafish oocyte. As mentioned earlier, the cistrons are susceptible to cold emphasis and undergo rebuff to dramatic alteration, PCR cistron look gives the clear image of whether or non the cistron is altered after cryopreservation. It is non known any difference between vitrification and decelerate chilling methods result in different consequences, nevertheless, as an initial effort the oocyte of zebrafish is extracted and utilizing methyl alcohol as a cryoprotectant, the oxidative emphasis if any induced for controlled slow chilling will be reviewed. However, vitrification requires utilizing high concentration of cryoprotectant, although, the intracellular ice is avoided there are plentifulness of hazard like osmotic daze and toxicity of cryoprotectant.
Viability trial of cryopreseved cells
The viability of the cells after cryopreservation is besides indispensable to describe the extent of oxidative emphasis. Most of the oocyte cryopreservation trials use staining as a standard viability trial. Despite of many standard dyes available for staining, the tryptan blue has become the most common dye used to measure the viability ( Chen et al. , 2006 ) . In peculiar, ovarian cerebral mantle staining with critical dye or fluorescent unrecorded or dead investigations. The advantage upon tissue warming follicles is released from cortical tissue by mechanical dissection or enzyme lysis gives easy entree to measure the viability. However, harmonizing to santos et Al. ( ) the viability position of the thawed preantral follicles instantly after cryopreservation could non be determined accurately.
Review of all the literature suggests the cryopreservation protocol requires specific attending about the metabolic stresses the cells undergo during cryopreservation. More concerns about loss of viability per centum because of the metabolic emphasis both during and after cryopreservation should be considered. It is non known in peculiar usage of one cryoprotectant may hold less impact over the other cryoprotectant. If the cryoprotectant produces less emphasis the successful decrease of the temperature below the glass passage temperature may non be achieved. Therefore, optimisation of cryopreservation protocols perchance to hold diminished emphasis and the extent to which it affect the overall cellular activity need to be analyzed. Although cryopreservation has become the everyday method for continuing the endangered species for figure of old ages, the possibilities of loss of its viability over the clip has to be major concern and emphatic surveies on such variables are extremely required.