The process of preserving the biological material using ultra low temperature is generic method with high success rate recently. The ability of preserving samples at nearly a frozen state is often referred to as cryobiology, and the evidences show history rolls back to B.C. The process of cryobiology involves lowering the temperature to liquid nitrogen temperature (-1960C) and subsequent thawing to room temperature when required (Gordan, 2003). This is because all the metabolic activity of the cells tends to stand still at ultra low temperature promising the integrity of the samples (Fuller 1991).
Although, success of preserving samples has been reported from -200C to -1960C, the technique of cryopreservation refers storing the biological materials at -1960C (Fuller, 1991). In all the cases, the designated temperature is either achieved by cooling the samples at slow rate and this process in controlled slow cooling or directly plunging in to liquid nitrogen (Borni, 2008). Attempts were also made to slow cool the temperature to higher negative temperature and then sudden plunging in to liquid nitrogen temperature (Borni, 2008).
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However, it has been recognized that the water is the primary component of living cells, the formation of ice is inevitable and the greatest challenge in cryopreservation is to avoid ice. When the system is cooled the formation of ice is unavoidable when the temperature lowers below the freezing point of water i.e. 00C (Speroff and Fritz, 2005). However, no living biologics seem to form ice at 0C because the water tends to super cool. Supercooling is an important phenomenon by which the water is cooled below its freezing point without forming ice (Gordan, 2003). In general water freezes only at -420C, the presence of heterogeneous ice nucleation (bacteria or bubbles) forms ice at higher temperature than actual water crystallization. When heterogeneous nucleation is absent the water itself forms ice at -400C because of homogenous nucleation point. The underlying principle in establishing successful protocols of cryopreservation is the taking the water out from the cells when cooling and redistribution of water to the cell when they are thawed. This can be achieved by suitable cryoprotectants supplied at correct concentration, which can act either from inside or outside the cells (Rall, 1993).
1.1 Glass Transition temperature
The glass transition temperature (Tg) is the exact temperature at which the water in the biologics is converted to rigid glass state. In strict sense, temperature slightly above the glass transition temperature may have slow chemical changes and therefore preserving the samples above Tg eventually degrade the stored samples (Nielsen and Landel, 1994).
The use of cryoprotectant lowers the water freezing at higher temperatures and water-cryoprotectant mixture eventually forms glass at glass transtition temperature instead of freezing at hetero/homogenous nucleation point (Fahy et al., 1984).
Cryoprotectants are the chemical substances that are often used to protect cells from damage during negative temperatures (Wolf, 1995). However, in order to achieve a glassy state of water the combination of high concentration of cryoprotectant(s) and an extremely rapid cooling is highly suggested (Vanderzwalmen et al., 2007). The selection of cryoprotectant therefore seems to be a crucial step in achieving the success in cryopreservation. The cryoprotectant lowers the equilibrium freezing point and lowers the homogenous ice nucleation point (Echlin, 1992). There are number of cryoprotectant available and they are generally grouped under two categories, 1) penetrating cryoprotectant and 2) non-penetrating cryoprotectant.
Glycerol is the best example of penetrating cryoprotectant. The name penetrating cryoprotectant is derived from the ability of the cryoprotectant to penetrate the plasma membrane. In addition, dimethyl sulphoxide (DMSO), methanol, ethanol, ethylene glycol, come under penetrating cryoprotectant. The great issue during selecting one of the cryoprotectant is the toxic effects. On the other hand, sucrose, polyethylene glycol (PEG) is the most extensively used non-penetrating cryoprotectant (Wishart G J, 2001). The non-penetrating cryoprotectant cannot enter the plasma membrane and therefore stays in the extracellular space. It is clearly revealed that the formation of ice in the extracellular space is inevitable when the temperature lowers below freezing point of water. The selection of cryoprotectant may also consider this phenomenon (Mazur, 1984).
1.3 Controlled slow cooling
Although, the technique of controlled slow cooling requires little or perhaps no cryoprotectant, the success of the technique relies greatly on the cryoprotective medium. For fish oocyte cryopreservation commonly used cryoprotectants such as methanol and glucose has been widely used (Guan et al., 2008). When the embryos are to be cryopreserved the cryoprotectant addition takes place either at room temperature of at 00C for about half an hour to one hour and this could be either one-step or multistep.
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The selection of cooling rate is an important factor in establishing the higher success of the fish oocyte viability. For embryos the cooling rate ranges from 0.01 to 0.750C/min. However, this traditional technique requires cryoprotectant loading and the osmotic removal of water during slow cooling (Hadedorn et al., 1998). Since most of the cryopreservation technique involves successful reduction of the temperature from physiological temperature to freezing point and the next step is the subsequent reduction of temperature to the liquid nitrogen temperature where the integrity of the samples is maintained.
The fish oocyte cryopreservation often encounters mechanical, thermal and chemical stresses. These injuries could damage the meiotic spindle and sometimes it may directly affect the zona pellucida (Baka et al., 1995). Such injury may affect the survival rate (Van der Elst, 2003). Chilling injury is one of the limiting factors for successful cryopreservation. The permanent damage occurs as a result of cooling the temperature to low without freezing temperatures is known as chilling injury (Ghetler et al., 2005). The membrane structure of the samples undergoing chilling injury is altered and hence the integrity of the samples is degraded (Zeron et al., 1999). In essence, the chilling injury seems to affect the oocyte microtubules (Alertini and Eppig, 1995). The chilling injury as it affects the membrane structures it tend to be a limiting factor in order to establish a thriving cryopreservation protocol. The chilling sensitivity of the oocytes increased upon increasing exposure time and the subzero temperature the oocytes are exposed to. The chilling sensitivity greatly varied between the oocytes containing yolk and without yolk (Vanderzwalmen,P. et al., 2007)
1.4 Aquatic species cryopreservation
The benefit of cryopreservation is the ability to bank the genetic material for numerous years at effectively low cost. There is a huge demand of fish all round the year and it has modernized the aquaculture industry (Bromage, 1995). Cryopreservation of such commercially essential fish embryos or eggs or sperms may yield high profit because the maintenance of fish in natural environment is costly, labour intensive and space consuming (Bart, 2000). Moreover, individual strains may encounter disease or natural disruption; In contrast, cryopreservation involves storage of samples and ensures contamination free germplasm (Rana, 1995). In addition, the cryopreservation of fish gametes guarantees the loss of fish species as it is globally threatened species (Guan et al., 2008). Moreover, preserving haploid nuclear genomes of viable male and female reproductive cells is the most consistent method for maintaining the count of the fishes. Current evidences suggest that cryopreserving fish eggs or embryos have been tried in more than 20 fish species.
The process of cryopreservation is not simply freezing samples, however it requires proper sample collection, refrigerated storage, and careful freezing without ice and thawing. The great barrier of fish cryopreservation is the small body size and less sperm volumes (Tiersch, 2001).
Plenty of cryopreservation studies have been attempted in fishes since Blaxter (1953) first published his report. Till date reports covers cryopreserving large body fish cultures, catfishes and carps (Scott and Baynes, 1980). The most studies focused on sperm cryopreservation and the sperm cryopreservation of fish has been successful in fish for over 80 species including fresh and salt water fish (Rana, 1995; Leung and Jaemison, 1991).
1.5 Zebrafish as a model
Recent studies stress the use of zebrafish (Danio rerio) as a best model after the prominent report published by Harvery et al. (1982)( Zhang,T et al., 2009). Although the successful cryopreservation of eggs, oocytes and ovarian tissues is still lacking with the fish species, zebrafish is the widely used sample for cryopreservation studies (Mazur et al., 2008). Zebrafish belongs to the family Cyprinidae and the nativity stretches to India and Pakistan. The reason for using zebrafish more often as in an experimental design particularly with egg cryopreservation is the production of fairly large amount of fertilized eggs (Selman et al., 1993).
In most of the fish studies, zebrafish is chosen because of the ease maintenance, prolific production of embryos, and their well-characterized, rapid development (Watersfield, 1983). In addition, small size and fecundity supports the easy storage of the fish for large scale studies. The zebrafish has become the most accepted model for the study of development and genetics. Moreover, the function of the most genes in the genome of the zebrafish is well established and the genome is fully mapped.
1.6 Ovary of zebrafish
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The maturation of the zebra fish oocyte can be grouped under five different stages as follows, The size of the stage I follicle was reported to be less than 0.14 mm in diameter, (the primary growth stage),whereas the size of stage II follicle ranges between a diameter of 0.14-0.34 mm diameter. (cortical alveoli stage),The oocyte grows up to more than 0.5 mm diameter at stage III (vitellogenic stage), stage IV (the so called maturation stage), stage V (mature egg) (Selman et al., 1993; Zampolla et al., 2008). All the stages of development consist of specific characteristics of membrane composition, protein and lipid content and organelles arrangement. This is considered to be the reason for variation in chilling injury for different stages of oocytes (Zampolla et al., 2008). Furthermore, the oocyte at early developmental stages cannot be cultured in vitro and this limits the possibilities of cryopreservation.
The oocytes at any stage can be separated with careful pipetting. Hankâ€™s solution along with simple actions from forceps were also the possible source of separating the oocyte (Adopted from Cryobiology practical session (2009) Available at www.breo.beds.ac.uk). Folliculogenesis and oogenesis comprises of the development of ovarian follicle, it also involves the complete process of meiosis, and the genetic coding from RNAâ€™s and protein. Most importantly, the acquired gene to withstand the embryo development until the stage where zygotic gene activation completes (Matova and Cooley, 2001).
In order to achieve successful cryopreservation protocol for oocyte cryopreservation, it is highly essential to consider the membrane permeability (Borni, 2008). In previous studies, three methods have been highly concentrated; this includes membrane permeability parameter estimation depending on oocyte volumetric changes. This is being measured with different concentration of cryoprotectant (Borni, 2008. It is also possible to assess the uptake by radio-labelled cryoprotectant (Ezcurra et al., 2009).
1.7 zebrafish oocyte cryopreservation
The cryopreservation of oocytes has specific advantages over embryo including the smaller size. Although, successful cryopreservation of mammalian species exist (Albertini et al., 1995), the fish oocytes and egg are more likely to contain yolk that is made up of proteins and lipids, and therefore the cryopreservation protocol is still not possible. Therefore, the cryopreservation studies conducted on embryo gives a good range of explanation for lack of successful cryopreservation of oocyte. Moreover, the chorion may be more permeable to water and solutes to exit and enter the cells. The ovarian follicles of the zebrafish are typically larger and the high yolk content often leads to difficulties in establishing successful cryopreservation protocols.
There are number of problems reported in the cryopreservation of the fish eggs and embryos including the barriers to water and cryoprotectant. The main issue to achieve the cryopreservation is the large size of the oocyte. This is believed to be a problem because the large size of oocyte might result in lower surface area to the volume ratio, this obviously affect the rate water and cryoprotectant movement in and out of the cells (Isayeva et al., 2004). The concerns of yolk with varied osmotic properties of plasma (Liu et al., 2001). The other difficulties often encountered are the chilling injury. However, the effect of chilling injury seems to be highly related to stage at which the oocytes are cryopreserved along with the temperature used and length of exposure to that particular temperature (Hagedorn et al., 1997; Zhang and Rawson, 1995). The chilling sensitivity seems also to be a problem (Hagedorn et al., 1997) especially in ovarian cryopreservation of zebrafish. Although, the use of cryoprotectant may be the best option available, more the concentration of cryoprotectant the lesser chances of chilling injury. However, increase in concentration increases the risk of toxicity (Leung, 1991).
Current evidences strongly suggest that viable eggs and embryos are preserved at -20Cand -55C respectively, and this seems to be achieved by using 8 to 14% DMSO (Erdahl and Graham, 1987)( Zhang,T., et al.,2005). Since the temperature was very high when compared with liquid nitrogen temperature that is often used for long term storage, it is not clearly known whether the tissues completely avoided ice. The chances of water existing in the supercooled state tend to be an interesting argument. This point of view, raises another question if the cryoprotectant used at different concentration actually permeated inside. Nevertheless, it has been found that cryoprotectant could penetrate zebrafish embryos, despite the penetration appears to be dissimilar because of the synctial layer of the yolk (YSL) and differentiating blastoderm cells (Hagedorn et al., 1997). In addition long term exposure of the embryos to cryoprotectants might be significantly toxic (Shafer, 1981). This is believed to happen because the cryoprotectant may possibly damage the cellular proteins (Harvey, 1982).
Cryopreserving the oocyte has much advantage including the smaller size, and since absence the chorion perhaps reduces the rate at which water and cryopretectant movement. Nevertheless, the oocytes has been demanded to undergo in vitro maturation, ovulation and fertilization, when cryopreservation is successful. Recent studies elucidate that the ovarian follicles are more permeable to water and the cryoprotectants used when compared with embryos (Zhang et al., 2005). In most instance, the oocyte cryopreservation of zebrafish refers to the ovarian follicles that consist of oocyte with its attached theca and granulose cells.
The gametes from the animals can be possibly obtained from undifferentiated progenitors of the germ-cell lineage. Eggs or sperms can be derived for the progenitor cells. Lot of studies underpin the chances of using microinjection into the peritoneal cavity of freshly hatched fish embryos.
1.8 Elucidation of viability after cryopreservation
As explained earlier, the cryopreservation of oocyte has not developed still, however, there has been methods developed to assess the viability of cryopreserved sample. It is fair to understand, that it would be important to understand the damage incurred by the cryopreservation process rather than counting viability (Nagy et al ., 2003).
There have been four methods developed to measure the viability. This includes trypan blue (TB) staining, thiazol MTT staining, flourescein diacetate staining and in vitro maturation followed by observation of germinal vesicle breakdown (GVBD) (Zhang and Lubzens, 2009). In TB staining method, the colouring agent is used to assess the cell membrane integrity. Generally, yellow coloured 3 or 4,5-Dimethylthazol-2yl-2,5-diphenyltetrazolium bromide will be reduced to purple coloured formazan. This conversion of tetrazole to the purple formazan usually takes place in mitochondria of living cells. However, there will be no reaction if the reductase enzyme in the mitochondria is inactive. Therefore, indicating the viability. There is an alternative dye that has been tested for assessing the viability. Diacetyl fluorescein which is a nonflourescent derivative of fluoresces in, may penetrate the cell membranes and becomes fluorescent after hydrolysis by esterases that are seen in the cytoplasm of viable cells (Mandelbaum, 1998). The so formed fluorescein can be dedected by using epifluorescent microscopy. Thus staining with both TB and fluorescein diacetate is merely simple (Zampolla et al., 2008).
In contrast, the assessment with viability using GVBD is restricted to oocytes at stage III. However, GVBD is quite possible to measure the viability at the oocytes developing later than stage III. Since this is a in vitro method, the assessment with TB was found to be more appropriate ( Plachinta,M.et al., 2004) (Zampolla et al., 2008).
1.9 Available cryopreservation attempts
Most of the previous studies mainly focused on controlled slow cooling (Plachinta et al., 2004). However, the most commonly used cryoprotectant for oocyte cryopreservation has been 4M methanol in addition with 0.2 M glucose. There has been a number of slow cooling rates being tested and it varied from 0.30C/min to 20C/min (Guan et al., 2008).
1.10 Aim of this study
In the present study, attempts will also be made to check the supercooling ability of oocytes. For this procedure two or three different cooling rates will be selected. The rate of interest is 0.30C/min, 20C/min and 2400C/min. This is hypothesised to be best done in two ways. In one method, cooling the oocytes, up to the supercooling limit and then sudden transfer of liquid nitrogen will be performed.
Further decline in fish species can be only controlled by cryopreserving the germ cells. Oocytes seem to have little advantage over the embryo both in terms of ethical and technical. Both vitrification and controlled slow cooling has been proved to be the best methods, none of the method look superior to other. More recently the cryopreservation attempts have been directed to use low toxicity cryoprotectant mixtures, and the focus has now terminated to protect the oocyte at sub-zero temperature.
The selection of either vitrification or controlled slow cooling is therefore essential for oocyte cryopreservation because the chilling injury has been a limiting factor. The sensitivity of the cells to low temperature is suggested to be highly related to the intraembryonic lipids and hence vitrification becomes an efficient method. However, the controlled slow cooling will also become an effective method, if the chilling sensitivity may be lowered by using suitable cryoprotectant. Careful slow cooling of the oocytes may have some benefits like penetration of cryoprotectants and reducing the chilling injury, whereas the sudden cooling by vitrification also avoids the intracellular ice formation because the rate of cooling is very rapid. This project is particularly designed to study if any of the methods has slight advantage/disadvantage over the other.