a normal cytoskeleton , composed of microfilaments , intermediate filaments , and microtubules , is critical for cell functions and embryo development . as dramatic as cell death , damage to the cytoskeleton renders the affected cell non - viable and the cell will not completely develop and survive . this is because the cytoskeleton gives strong support to the cell plasma membrane and maintains intracellular organelle organization . due to its interaction with the cell membrane , the cytoskeleton may also have a significant role in determining post - dehydration and post - thaw viability . it is capable of modifying membrane structure , altering shape or morphology of the membrane , changing the mechanical properties of the cell , and influencing the membrane water transport . cytoskeletal alterations in cryopreserved embryos are attributed to many confounding factors such as developmental stage during cryopreservation , the origin of embryos ( in vivo or in vitro ) , the type of cryoprotectant , selection of cryopreservation methods , and degree of zona absence . therefore , any alterations and disruptions in cytoskeleton may explain most of the detrimental effects in cell morphology and organelle distributions of any freezing - induced methods . the storage of gametes could provide a powerful tool in research and contributes to advances in mammalian infertility treatment , the conservation of endangered species , and reproductive biology , as well as increasing the availability of oocytes for research applications such as genetic engineering or embryo cloning . therefore , cryopreservation of oocytes and embryos is an integral part of assisted reproductive technology ( art ) . during the past few decades , it is important to study the ultrastructural damage of the embryos during cryopreservation because the ultrastructural components of mouse embryos undergo dramatic architectural changes , considerable stress , and morphological and functional damage during cryopreservation . in many cases , the damage can reduce embryo viability due to destabilization and disruption of cell organelles   and the cytoskeleton . cryopreservation may also cause injuries to the cell , such as mitochondria and endoplasmic reticulum alterations , poorly developed desmosomes , and lack of tight junctions . therefore , by understanding the type of cytoskeletal damage from vitrification and slow freezing , new strategies might be developed and implemented to improve cryopreservation techniques . embryos can be damaged during cryopreservation and thawing , either by the formation of large intracellular ice crystals or by increased intracellular concentration of solutes and accompanying changes that result from dehydration of cells during cryopreservation . damage may occur to embryos when they pass through a range of temperatures during cryopreservation . early injuries may start at between + 15c to 5c when the meiotic spindles , microtubules , and cytoplasmic lipid will be destroyed by the chilling effects . when the temperatures goes down to 5c to 80c , the formation of extracellular and intracellular ice crystals are the major cause of embryo damage . when the temperature goes to 150c , fracture of the zona pellucida can not be avoided . actin treadmilling provides a signal modulating nuclear gene expression and provides an additional regulatory twist to cell motility . actin has been recently proposed to be an important factor throughout the entire rna biogenesis pathway as a component of chromatin remodeling complexes . during morphogenesis , actin plays a major role in cellular movements , including ingression , epiboly , invagination , involution , and delamination . . therefore , any changes and disruption in actin structures will jeopardize the survival of embryos , perhaps because the cryopreservation process may cause actin depolymerization that completely blocks chromosome migration . recent improvements of florescence dyes and microscopy techniques allow detailed examination of specific cellular structures as microfilaments , microtubules , mitochondria , zona membranes , and nuclei . a confocal laser scanning microscope can be used to improve the selection of the most viable embryos by detecting the degree of cytoskeletal and ultrastructural damage of the embryos following cryopreservation . therefore , the aim of this study was to document cellular damage after cryopreservation of embryos using slow freezing and vitrification and to evaluate the cellular sensitivity to cryopreservation . information on cytoskeletal damage may provide a good indication of cryotolerance and enhances the efficiency of embryonic cryopreservation . fifty female icr mice aged 46 weeks and weighing 2530 grams were used in this study . all animals were kept in standard laboratory conditions at 27c with 12-h light - dark periods and they were given food pellets and water ad libitum . ethics approval from the university animal care and use committee ( code : acuc-7/11 ) was obtained and all procedures used followed strictly the malaysian animal ethics guidelines . female mice were superovulated using pregnant mare s serum gonadotropin ( pmsg ) ( 5 iu / kg b.w . ) and human chorionic gonadotropin ( hcg ) ( 5 iu / kg b.w . ) hormones [ intervet , holland ] intraperitoneally 48 h later before being mated with the fertile male mice with a ratio of 1:1 . female mice with the presence of vaginal plugs were considered to be pregnant and were selected as embryo donors . ovaries from each animal were placed into the petri dishes containing m2 medium [ sigma , usa ] , fallopian tubes were excised , and embryos were flushed under a dissecting microscope [ leica zoom 2000 , japan ] . only good quality 2-cell stage embryos with an intact zona pellucida and symmetrical blastomeres were selected for the experiments . the selected 2-cell embryos were rinsed with m2 medium and cultured in vitro in 24-well plates [ orange scientific , belgium ] filled with 100 l of m16 media [ sigma , usa ] overlaid with mineral oil [ sigma , usa ] to further develop to 4- and 8-cell stages . embryo culture was equilibrated in a water - jacketed 37c co2 incubator ( 5% co2 ; 95% air ) [ memmert , germany ] and the development of embryos was observed daily under the inverted microscope [ olympus 1x81 sf-3 , japan ] . embryos were cryopreserved at the 2- , 4- , and 8-cell stages of development . in this experiment , a polymer called efs40 was used as a cryoprotectant [ 18,3234 ] . a boat made from a styrofoam box with 1-cm thickness and grooves at least 5 cm height of liquid nitrogen was filled in the styrofoam box 30 min before the start of the experiment . embryos were collected using m2 media and kept at room temperature . a 1-ml syringe fitted with a white pipette tip thirty microliters of efs40 , corresponding to 15 ml of solution , was pulled in , followed by a 5-mm air space . then an additional 5 mm of solution was pulled in just after the air bubble near the opening . the straw was laid down carefully and a total of 10 embryos were aspirated in a minimal volume of m2 using a transfer pipette . the transfer pipette was inserted into the horizontally held straw and the embryos were expelled into the efs40 , while smoothly withdrawing the pipette . after that , the straw was sealed at the open end by using polyvinyl alcohol ( pva ) powder and was wiped clean . once the embryos were equilibrated with the cryoprotectant for 1 min at room temperature , the straw was placed on the groove in the styrofoam boat floating on liquid nitrogen vapor . after that , it was immersed in liquid nitrogen and stored in a liquid nitrogen tank until use . during the warming procedure , the straw was removed from the liquid nitrogen and then placed on the styrofoam boat . after being held in air , it was immersed in a 20c water bath for 60 s. the straws were then removed from the water bath and wiped dry . the seal was cut and the contents expelled into a dish containing 0.5 m sucrose . the embryos were then transferred into m2 media , rinsed twice in fresh m2 media , and placed in a m16 droplet overlaid with mineral oil for culture . the embryos were also cryopreserved at 2- , 4- , and 8-cell stages of development . in this experiment , liquid nitrogen and a boat made from a 1-cm  thick styrofoam box embryos were then aspirated into the straw containing d - pbs and dmso before being transferred into an ice bath for 30 min . the straw was then transferred to a 20c freezer for 30 min before being placed in a 80c freezer for 1 h. after that , the straw was placed on a styrofoam boat in liquid nitrogen vapor . after 30 min , the straw was immersed and stored in liquid nitrogen until use . to thaw the embryos following slow freezing procedures , the straws containing embryos were removed from the liquid nitrogen tank and then placed on a styrofoam boat for 15 min . when completely thawed , the seal was cut and the contents expelled into a dish containing d - pbs for washing . the embryos were then transferred into m2 media , rinsed twice with fresh m2 media and finally placed in m16 droplets covered with mineral oil for culture . the embryos were fixed with 4% paraformaldehyde for 24 h and the nuclei were stained with 4,6-diamino-2-phenylindole dihydrochloride ( dapi ) for 40 min before being permeabilized with 1% triton - x in phosphate buffer saline ( pbs ) . after 10 min incubation in 1% triton - x , actin structures were labelled with alexa fluor 635 and tubulin structures were labelled with anti--tubulin for 1 h , the embryos were washed with pbs twice for 10 min and counterstained with dapi for 40 min . the embryos were mounted on slides , sealed , stored in the dark at 4c overnight and finally viewed under a confocal laser scanning microscope ( clsm ) [ leica tcs sp5 aobs , germany ] . images taken from clsm were converted to jpeg format before being analyzed using qwin software v.3 . assessment of cytoskeletal quality and cellular damage are based on the intensity of fluorescent probes after immunofluorescence staining and the morphology of the embryos . the percentage of embryo development until the hatched blastocyst indicates the survivability of the embryos . intensities of fluorescent probes in each stage of embryos were compared between experimental groups using one - way anovas with a bonferroni correction . in all cases , statistics were performed using statistical package for social sciences version 16 ( spss inc , usa ) , and differences were considered significant when p<0.05 . fifty female icr mice aged 46 weeks and weighing 2530 grams were used in this study . all animals were kept in standard laboratory conditions at 27c with 12-h light - dark periods and they were given food pellets and water ad libitum . ethics approval from the university animal care and use committee ( code : acuc-7/11 ) was obtained and all procedures used followed strictly the malaysian animal ethics guidelines . female mice were superovulated using pregnant mare s serum gonadotropin ( pmsg ) ( 5 iu / kg b.w . ) and human chorionic gonadotropin ( hcg ) ( 5 iu / kg b.w . ) hormones [ intervet , holland ] intraperitoneally 48 h later before being mated with the fertile male mice with a ratio of 1:1 . female mice with the presence of vaginal plugs were considered to be pregnant and were selected as embryo donors . ovaries from each animal were placed into the petri dishes containing m2 medium [ sigma , usa ] , fallopian tubes were excised , and embryos were flushed under a dissecting microscope [ leica zoom 2000 , japan ] . only good quality 2-cell stage embryos with an intact zona pellucida and symmetrical blastomeres were selected for the experiments . the selected 2-cell embryos were rinsed with m2 medium and cultured in vitro in 24-well plates [ orange scientific , belgium ] filled with 100 l of m16 media [ sigma , usa ] overlaid with mineral oil [ sigma , usa ] to further develop to 4- and 8-cell stages . embryo culture was equilibrated in a water - jacketed 37c co2 incubator ( 5% co2 ; 95% air ) [ memmert , germany ] and the development of embryos was observed daily under the inverted microscope [ olympus 1x81 sf-3 , japan ] . embryos were cryopreserved at the 2- , 4- , and 8-cell stages of development . in this experiment , a polymer called efs40 was used as a cryoprotectant [ 18,3234 ] . a boat made from a styrofoam box with 1-cm thickness and grooves at least 5 cm height of liquid nitrogen was filled in the styrofoam box 30 min before the start of the experiment . embryos were collected using m2 media and kept at room temperature . a 1-ml syringe fitted with a white pipette tip thirty microliters of efs40 , corresponding to 15 ml of solution , was pulled in , followed by a 5-mm air space . then an additional 5 mm of solution was pulled in just after the air bubble near the opening . the straw was laid down carefully and a total of 10 embryos were aspirated in a minimal volume of m2 using a transfer pipette . the transfer pipette was inserted into the horizontally held straw and the embryos were expelled into the efs40 , while smoothly withdrawing the pipette . after that , the straw was sealed at the open end by using polyvinyl alcohol ( pva ) powder and was wiped clean . once the embryos were equilibrated with the cryoprotectant for 1 min at room temperature , the straw was placed on the groove in the styrofoam boat floating on liquid nitrogen vapor . after that , it was immersed in liquid nitrogen and stored in a liquid nitrogen tank until use . during the warming procedure , the straw was removed from the liquid nitrogen and then placed on the styrofoam boat . after being held in air , it was immersed in a 20c water bath for 60 s. the straws were then removed from the water bath and wiped dry . the seal was cut and the contents expelled into a dish containing 0.5 m sucrose . the embryos were then transferred into m2 media , rinsed twice in fresh m2 media , and placed in a m16 droplet overlaid with mineral oil for culture . the methods of slow freezing by nagy et al . ( 2003 ) were used with some modifications . the embryos were also cryopreserved at 2- , 4- , and 8-cell stages of development . in this experiment , liquid nitrogen and a boat made from a 1-cm  thick styrofoam box embryos were then aspirated into the straw containing d - pbs and dmso before being transferred into an ice bath for 30 min . the straw was then transferred to a 20c freezer for 30 min before being placed in a 80c freezer for 1 h. after that , the straw was placed on a styrofoam boat in liquid nitrogen vapor . after 30 min , the straw was immersed and stored in liquid nitrogen until use . to thaw the embryos following slow freezing procedures , the straws containing embryos were removed from the liquid nitrogen tank and then placed on a styrofoam boat for 15 min . when completely thawed , the seal was cut and the contents expelled into a dish containing d - pbs for washing . the embryos were then transferred into m2 media , rinsed twice with fresh m2 media and finally placed in m16 droplets covered with mineral oil for culture . the embryos were fixed with 4% paraformaldehyde for 24 h and the nuclei were stained with 4,6-diamino-2-phenylindole dihydrochloride ( dapi ) for 40 min before being permeabilized with 1% triton - x in phosphate buffer saline ( pbs ) . after 10 min incubation in 1% triton - x , actin structures were labelled with alexa fluor 635 and tubulin structures were labelled with anti--tubulin for 1 h , the embryos were washed with pbs twice for 10 min and counterstained with dapi for 40 min . the embryos were mounted on slides , sealed , stored in the dark at 4c overnight and finally viewed under a confocal laser scanning microscope ( clsm ) [ leica tcs sp5 aobs , germany ] . images taken from clsm were converted to jpeg format before being analyzed using qwin software v.3 . assessment of cytoskeletal quality and cellular damage are based on the intensity of fluorescent probes after immunofluorescence staining and the morphology of the embryos . the percentage of embryo development until the hatched blastocyst indicates the survivability of the embryos . intensities of fluorescent probes in each stage of embryos were compared between experimental groups using one - way anovas with a bonferroni correction . in all cases , statistics were performed using statistical package for social sciences version 16 ( spss inc , usa ) , and differences were considered significant when p<0.05 . the changes in actin and tubulin integrity in preimplantation embryos with and without prior cryopreservation were observed by using clsm . 2- , 4- , and 8-cell stage embryos  were chosen for the comparison between vitrification , slow freezing , and non - cryopreserved embryos . confocal images of embryos were analyzed by using qwin software v3 and the images are shown in figure 1a1i . figure 1a1c shows the controls ( non - cryopreserved ) embryos in 2- , 4- , and 8-cell stages . the vitrified embryos are shown in figure 1d1f and figure 1g1i shows the slow - frozen embryos in various developmental stages . red color represents the actin structures of embryos stained with alexa fluor 635 phalloidin , green stain of anti--tubulin represents the tubulin structures , and the nucleus of embryos appeared blue in color when it stained with dapi . fluorescence micrographs from figure 1a1i show the distribution of tubulin , actin , and nucleus clustering in controls , vitrified and slow - frozen , of 2- , 4- , and 8-cell stage embryos . the nuclei of individual cells were located in the center region of the cytoplasm of controls 2- and 4-cell ( figure 1a , 1b ) , as well as in vitrified 2- and 4-cell embryos ( figure 1d , 1e ) . outward migration of the nucleus of the blastomere is observed in the control 8-cell embryos ( figure 1c ) . however , major disruption of the nucleus occurred in vitrified 8-cell embryos ( figure 1f ) and all slow - frozen embryos ( figure 1g1i ) . complete perinuclear clustering of tubulin with a thin green stain were observed in control embryos ( figure 1a1c ) . in 8-cell embryos , the cortex area became strongly stained with anti--tubulin and perinuclear clustering was still observed . in the vitrified groups ( figure 1d , 1e ) , the embryos display partial migration of tubulin into the subcortical region because of the deleterious effect of vitrification . however , more damage in tubulin was observed in the 8-cell stage ( figure 1f ) . sizes of the perivitelline space were also increased in vitrified 2-and 4-cell embryos ( figure 1d , 1e ) as well as in slow - frozen 2-cell embryos ( figure 1 g ) . however , the fluorescent intensity seemed to decline slightly in all cryopreserved embryos ( figure 1d1i ) and the most damage were observed in vitrified and slow - frozen 8-cell stage embryos ( figure 1f and 1i ) . percentage of probes intensities in each confocal image of control and cryopreserved embryos were calculated using qwin software and the results are summarized in table 1 and figures 24 . table 1 shows percentage of fluorescent intensities ( % ) of tubulin , actin , and nucleus in 2- , 4- , and 8-cell stages of the preimplantation embryos following different cryopreservation ( vitrification or slow freezing ) . in all cryopreserved groups , as the number of the cells increased , fluorescent intensities of tubulin , actin , and nucleus were decreased slightly as compared to non - vitrified ( control ) groups . the results clearly show that both cryopreservation methods caused highly significant changes in cytoskeletal structures of embryos , especially in tubulin , actin , and nucleus as compared to non - vitrified embryos ( p<0.001 ) . however , intensity comparisons showed that actin , tubulin , and nucleus intensities of vitrified embryos were slightly higher than in slow - frozen embryos . percentage of tubulin , actin , and nucleus were also compared between each developmental stage and freezing techniques . results show that vitrification methods caused highly significant changes in tubulin structures between 2- , 4- , and 8-cell stages ( p<0.001 ) . the percentages of intensities were also having significant difference between stages of development . however , the results were not significant between 4-cell and 8-cell stages in terms of nucleus intensities . figure 2 shows the percentage of tubulin intensities in control , vitrified , and slow - frozen groups . in the control ( non - vitrified ) group , the distributions of tubulin were increased as the number of the cells increased . the percentage of tubulin intensities in 2- , 4- , and 8-cell stage embryos were 49.51.5% , 69.42.4% , and 76.52.0% , respectively . this pattern also was seen in the vitrified and slow - frozen embryos but the increment was less than in the control embryos . in the vitrified group , the percentage of tubulin intensities were 21.81.0% , 34.81.0% , and 42.91.2% , respectively . the lowest intensities of tubulin were in the slow freezing group  11.30.5% , 11.20.6% , and 19.90.9% , respectively . figure 3 shows the percentage of actin intensities in control , vitrified , and slow - freezing groups . as shown in the graph , the percentage of actin intensities in 2- , 4- , and 8-cell stages were 28.11.6% , 32.51.1% , and 37.20.9% , respectively . as seen in tubulin , the pattern of the actin intensity increased from 2- to 8-cell stage but there was more damage in the 4-cell stage in all treatment groups . however , during the 8-cell stage , the actin intensity continued to increase . in the vitrified group , the actin intensities in 2- , 4- , and 8-cell stages were 6.30.3% , 16.01.0% , and 13.70.5% , respectively . for the slow - freezing group , the intensity of actin was 6.20.2% in 2-cell stage and it decreased to 4.20.3% in the 4-cell stage , and then increased again in the 8-cell stage to 9.10.3% . figure 4 shows the percentage of nucleus intensities in control , vitrified , and slow - frozen groups . from the results of this study it can be concluded that the most damage is seen in the nucleus after vitrification and slow freezing . as the number of the cells increased , nuclear intensity increased , as observed with the intensities of tubulin and actin . intensities of nuclei in the control group were 50.01.7% , 52.01.4% , and 49.12.1% , respectively . the intensities were decreased in the vitrified group  6.60.4% , 14.00.7% , and 14.60.6% . however , the lowest intensities were in the slow - frozen embryos as compared to the normal and vitrified embryos  3.60.3% , 3.80.3% , and 9.30.3% , respectively . the changes in actin and tubulin integrity in preimplantation embryos with and without prior cryopreservation were observed by using clsm . 2- , 4- , and 8-cell stage embryos  were chosen for the comparison between vitrification , slow freezing , and non - cryopreserved embryos . confocal images of embryos were analyzed by using qwin software v3 and the images are shown in figure 1a1i . figure 1a1c shows the controls ( non - cryopreserved ) embryos in 2- , 4- , and 8-cell stages . the vitrified embryos are shown in figure 1d1f and figure 1g1i shows the slow - frozen embryos in various developmental stages . red color represents the actin structures of embryos stained with alexa fluor 635 phalloidin , green stain of anti--tubulin represents the tubulin structures , and the nucleus of embryos appeared blue in color when it stained with dapi . fluorescence micrographs from figure 1a1i show the distribution of tubulin , actin , and nucleus clustering in controls , vitrified and slow - frozen , of 2- , 4- , and 8-cell stage embryos . the nuclei of individual cells were located in the center region of the cytoplasm of controls 2- and 4-cell ( figure 1a , 1b ) , as well as in vitrified 2- and 4-cell embryos ( figure 1d , 1e ) . outward migration of the nucleus of the blastomere is observed in the control 8-cell embryos ( figure 1c ) . however , major disruption of the nucleus occurred in vitrified 8-cell embryos ( figure 1f ) and all slow - frozen embryos ( figure 1g1i ) . complete perinuclear clustering of tubulin with a thin green stain were observed in control embryos ( figure 1a1c ) . in 8-cell embryos , the cortex area became strongly stained with anti--tubulin and perinuclear clustering was still observed . in the vitrified groups ( figure 1d , 1e ) , the embryos display partial migration of tubulin into the subcortical region because of the deleterious effect of vitrification . however , more damage in tubulin was observed in the 8-cell stage ( figure 1f ) . sizes of the perivitelline space were also increased in vitrified 2-and 4-cell embryos ( figure 1d , 1e ) as well as in slow - frozen 2-cell embryos ( figure 1 g ) . however , the fluorescent intensity seemed to decline slightly in all cryopreserved embryos ( figure 1d1i ) and the most damage were observed in vitrified and slow - frozen 8-cell stage embryos ( figure 1f and 1i ) . percentage of probes intensities in each confocal image of control and cryopreserved embryos were calculated using qwin software and the results are summarized in table 1 and figures 24 . table 1 shows percentage of fluorescent intensities ( % ) of tubulin , actin , and nucleus in 2- , 4- , and 8-cell stages of the preimplantation embryos following different cryopreservation ( vitrification or slow freezing ) . in all cryopreserved groups , as the number of the cells increased , fluorescent intensities of tubulin , actin , and nucleus were decreased slightly as compared to non - vitrified ( control ) groups . the results clearly show that both cryopreservation methods caused highly significant changes in cytoskeletal structures of embryos , especially in tubulin , actin , and nucleus as compared to non - vitrified embryos ( p<0.001 ) . however , intensity comparisons showed that actin , tubulin , and nucleus intensities of vitrified embryos were slightly higher than in slow - frozen embryos . percentage of tubulin , actin , and nucleus were also compared between each developmental stage and freezing techniques . results show that vitrification methods caused highly significant changes in tubulin structures between 2- , 4- , and 8-cell stages ( p<0.001 ) . the percentages of intensities were also having significant difference between stages of development . however , the results were not significant between 4-cell and 8-cell stages in terms of nucleus intensities . figure 2 shows the percentage of tubulin intensities in control , vitrified , and slow - frozen groups . in the control ( non - vitrified ) group , the distributions of tubulin were increased as the number of the cells increased . the percentage of tubulin intensities in 2- , 4- , and 8-cell stage embryos were 49.51.5% , 69.42.4% , and 76.52.0% , respectively . this pattern also was seen in the vitrified and slow - frozen embryos but the increment was less than in the control embryos . in the vitrified group , the percentage of tubulin intensities were 21.81.0% , 34.81.0% , and 42.91.2% , respectively . the lowest intensities of tubulin were in the slow freezing group  11.30.5% , 11.20.6% , and 19.90.9% , respectively . figure 3 shows the percentage of actin intensities in control , vitrified , and slow - freezing groups . as shown in the graph , the percentage of actin intensities in 2- , 4- , and 8-cell stages were 28.11.6% , 32.51.1% , and 37.20.9% , respectively . as seen in tubulin , the pattern of the actin intensity increased from 2- to 8-cell stage but there was more damage in the 4-cell stage in all treatment groups . however , during the 8-cell stage , the actin intensity continued to increase . in the vitrified group , the actin intensities in 2- , 4- , and 8-cell stages were 6.30.3% , 16.01.0% , and 13.70.5% , respectively . for the slow - freezing group , the intensity of actin was 6.20.2% in 2-cell stage and it decreased to 4.20.3% in the 4-cell stage , and then increased again in the 8-cell stage to 9.10.3% . figure 4 shows the percentage of nucleus intensities in control , vitrified , and slow - frozen groups . from the results of this study it can be concluded that the most damage is seen in the nucleus after vitrification and slow freezing . as the number of the cells increased , nuclear intensity increased , as observed with the intensities of tubulin and actin . intensities of nuclei in the control group were 50.01.7% , 52.01.4% , and 49.12.1% , respectively . the intensities were decreased in the vitrified group  6.60.4% , 14.00.7% , and 14.60.6% . however , the lowest intensities were in the slow - frozen embryos as compared to the normal and vitrified embryos  3.60.3% , 3.80.3% , and 9.30.3% , respectively . the changes in actin and tubulin integrity in preimplantation embryos with and without prior cryopreservation were observed by using clsm . 2- , 4- , and 8-cell stage embryos  were chosen for the comparison between vitrification , slow freezing , and non - cryopreserved embryos . confocal images of embryos were analyzed by using qwin software v3 and the images are shown in figure 1a1i . figure 1a1c shows the controls ( non - cryopreserved ) embryos in 2- , 4- , and 8-cell stages . the vitrified embryos are shown in figure 1d1f and figure 1g1i shows the slow - frozen embryos in various developmental stages . red color represents the actin structures of embryos stained with alexa fluor 635 phalloidin , green stain of anti--tubulin represents the tubulin structures , and the nucleus of embryos appeared blue in color when it stained with dapi . fluorescence micrographs from figure 1a1i show the distribution of tubulin , actin , and nucleus clustering in controls , vitrified and slow - frozen , of 2- , 4- , and 8-cell stage embryos . the nuclei of individual cells were located in the center region of the cytoplasm of controls 2- and 4-cell ( figure 1a , 1b ) , as well as in vitrified 2- and 4-cell embryos ( figure 1d , 1e ) . outward migration of the nucleus of the blastomere is observed in the control 8-cell embryos ( figure 1c ) . however , major disruption of the nucleus occurred in vitrified 8-cell embryos ( figure 1f ) and all slow - frozen embryos ( figure 1g1i ) . complete perinuclear clustering of tubulin with a thin green stain were observed in control embryos ( figure 1a1c ) . in 8-cell embryos , the cortex area became strongly stained with anti--tubulin and perinuclear clustering was still observed . in the vitrified groups ( figure 1d , 1e ) , the embryos display partial migration of tubulin into the subcortical region because of the deleterious effect of vitrification . however , more damage in tubulin was observed in the 8-cell stage ( figure 1f ) . sizes of the perivitelline space were also increased in vitrified 2-and 4-cell embryos ( figure 1d , 1e ) as well as in slow - frozen 2-cell embryos ( figure 1 g ) . however , the fluorescent intensity seemed to decline slightly in all cryopreserved embryos ( figure 1d1i ) and the most damage were observed in vitrified and slow - frozen 8-cell stage embryos ( figure 1f and 1i ) . percentage of probes intensities in each confocal image of control and cryopreserved embryos were calculated using qwin software and the results are summarized in table 1 and figures 24 . table 1 shows percentage of fluorescent intensities ( % ) of tubulin , actin , and nucleus in 2- , 4- , and 8-cell stages of the preimplantation embryos following different cryopreservation ( vitrification or slow freezing ) . in all cryopreserved groups , as the number of the cells increased , fluorescent intensities of tubulin , actin , and nucleus were decreased slightly as compared to non - vitrified ( control ) groups . the results clearly show that both cryopreservation methods caused highly significant changes in cytoskeletal structures of embryos , especially in tubulin , actin , and nucleus as compared to non - vitrified embryos ( p<0.001 ) . however , intensity comparisons showed that actin , tubulin , and nucleus intensities of vitrified embryos were slightly higher than in slow - frozen embryos . percentage of tubulin , actin , and nucleus were also compared between each developmental stage and freezing techniques . results show that vitrification methods caused highly significant changes in tubulin structures between 2- , 4- , and 8-cell stages ( p<0.001 ) . the percentages of intensities were also having significant difference between stages of development . however , the results were not significant between 4-cell and 8-cell stages in terms of nucleus intensities . figure 2 shows the percentage of tubulin intensities in control , vitrified , and slow - frozen groups . in the control ( non - vitrified ) group , the distributions of tubulin were increased as the number of the cells increased . the percentage of tubulin intensities in 2- , 4- , and 8-cell stage embryos were 49.51.5% , 69.42.4% , and 76.52.0% , respectively . this pattern also was seen in the vitrified and slow - frozen embryos but the increment was less than in the control embryos . in the vitrified group , the percentage of tubulin intensities were 21.81.0% , 34.81.0% , and 42.91.2% , respectively . the lowest intensities of tubulin were in the slow freezing group  11.30.5% , 11.20.6% , and 19.90.9% , respectively . figure 3 shows the percentage of actin intensities in control , vitrified , and slow - freezing groups . as shown in the graph , the percentage of actin intensities in 2- , 4- , and 8-cell stages were 28.11.6% , 32.51.1% , and 37.20.9% , respectively . as seen in tubulin , the pattern of the actin intensity increased from 2- to 8-cell stage but there was more damage in the 4-cell stage in all treatment groups . however , during the 8-cell stage , the actin intensity continued to increase . in the vitrified group , the actin intensities in 2- , 4- , and 8-cell stages were 6.30.3% , 16.01.0% , and 13.70.5% , respectively . for the slow - freezing group , the intensity of actin was 6.20.2% in 2-cell stage and it decreased to 4.20.3% in the 4-cell stage , and then increased again in the 8-cell stage to 9.10.3% . figure 4 shows the percentage of nucleus intensities in control , vitrified , and slow - frozen groups . from the results of this study it can be concluded that the most damage is seen in the nucleus after vitrification and slow freezing . as the number of the cells increased , nuclear intensity increased , as observed with the intensities of tubulin and actin . intensities of nuclei in the control group were 50.01.7% , 52.01.4% , and 49.12.1% , respectively . the intensities were decreased in the vitrified group  6.60.4% , 14.00.7% , and 14.60.6% . however , the lowest intensities were in the slow - frozen embryos as compared to the normal and vitrified embryos  3.60.3% , 3.80.3% , and 9.30.3% , respectively . in this study , the cytoskeletal organizations of the preimplantation murine embryos following different types of cryopreservation were observed . the results from confocal images clearly showed that cryopreservation by using vitrification and slow freezing methods caused significant percentage changes in fluorescent intensity of tubulin , actin , and nucleus integrity as compared to non - vitrified embryos ( p<0.001 ) . however , intensity comparisons showed vitrified embryos have higher fluorescent intensity in cytoskeletal organization as compared to slow - frozen embryos . the prominent changes in cytoskeletal organizations seen in slow freezing and vitrification may be due to the changes in metabolic function , developmental capacity , physiological necessities , and requirements of the embryos . the cytoskeleton of a mammalian embryo is made of a unique and fragile intracellular network of microfilaments and microtubules that play major roles in normal function and development of the cells . the cytoskeleton also plays a significant role in determining the cell s response to freeze / thaw and dehydration stresses , and cell survival . an intact cytoskeleton is also essential for cytokinesis and karyokinesis ; therefore , if the cytoskeleton is irreversibly damaged , the mitotic cell cycle will end , rendering the affected cell non - viable . results from this study demonstrate that cryopreservation using vitrification and slow freezing methods caused deleterious damage in the embryo cytoskeleton , such as actin , tubulin , and nucleus at various developmental stages of preimplantation embryos as compared to non - cryopreserved embryos . ( 2008 ) , who found that the quality and distribution of the cytoskeleton was affected by the freezing technique employed and the embryo age / size . they also found that the effect was significantly lower in vitrified and conventionally frozen / thawed embryos , as compared to controls . microfilaments and microtubules are very sensitive to the cooling process [ 3840 ] , and their integrity is essential for proper progression through meiosis . in fact , tubulin is responsible for the constitution of the meiotic spindle and drives the proper alignment of chromosomes on the metaphase plate , and microfilaments are necessary for cortical granule migration and polar body expulsion . the degree of repolymerization was critically dependent on the method used to dilute or remove the cpa , and failure to completely rehydrate resulted in an embryo with pyknotic nuclei and both a diminished magnitude and altered cellular distribution of microfilaments and microtubules . in the present study , consists of both permeating and non - permeating cryoprotectant and it is used to cause a shift in the isotonic state between the intracellular and extracellular spaces , driving water out of the cell . permeating cryoprotectant produces large - volume changes during freezing and thawing processes . therefore , less damage occurred in vitrified embryos compared to slow - frozen embryos as the results of cryoprotective effect of efs40 . cells are protected by permeating agents such as eg and dmso , which are organic solutes , during cooling and warming procedures prior to and after storage in liquid nitrogen . our findings shows that vitrification by using efs40 as cpas enhanced and gave better protection to 2- and 4-cell embryos in terms of cytoskeletal damage and embryo quality as compared to slow freezing . these findings confirmed that low toxicity of efs40 is suitable to vitrify embryos , as reported by chen et al . this finding also agrees with nowshari and brem ( 2001 ) , who reported that eg - based solutions were the best cryoprotectant for embryo cryopreservation . ( 2001 ) also found that inclusion of polymers such as ficoll and sucrose improved embryo survivability after freezing . this may be because the amount of toxic cryoprotectants may be decreased by replacing some cryoprotectants with polymers and sugars . ficoll has been used successfully with efs40 solutions during cryopreservation and researchers found that it is suitable for cryopreservation of 8-cell and morula stage mouse embryos [ 4547 ] and hatched blastocysts . vitrification leads to an extreme increase in viscosity in the presence of high concentration of cpas , permitting the transition of aqueous solutions into a glassy state , bypassing the crystalline state . the concentration of cpas required for vitrification has been calculated as being 47 times higher than that required in slow freezing , and may result in either osmotic or chemical toxicity . this stage - dependant sensitivity of embryos to damage during cryopreservation , and differences between species and freezing techniques , are major obstacles to the widespread application of cryopreservation in commercial practice . our study focused on the effect of vitrification and slow freezing in early preimplantation stages ( 2- , 4- , and 8-cell ) of mouse embryos , as other researchers have demonstrated that early stage embryos can be vitrified [ 5456 ] . however , cseh et al . ( 1999 ) claimed that the most suitable developmental stage for vitrification was the compacted morulae . in this case , the present study is limited because cytoskeletal images of embryos at morulae and blastocyst stages can not be analyzed by clsm due to overlapping of the fluorescent signals . this limitation was also found in a previous study in which researchers had difficulty identifying the distribution of the cytoskeleton within each blastomere because the fluorescence signals overlapped . our results show that cryopreservation using slow freezing methods have more deleterious effects on the cytoskeletal structures . our finding is in agreement with that of others who demonstrated that conventional slow freezing methods lead to moderate cytoskeleton disruption and cell death . dobrinsky et al . ( 2000 ) reported that vitrifying pig embryos caused considerable microfilament disruption and significantly reduced developmental competence . ( 2009 ) who showed that the slow freezing method used on feline embryos preserves the cytoskeleton organization better than vitrification . other factors such as physical injuries by intracellular and extracellular ice formation , cryoprotectant toxicity , osmotic stress , and chilling injuries have been proposed to contribute to embryo damage after cryopreservation . the formation of ice crystals that occurs in slow freezing methods disrupts the structure of the embryos during cryopreservation . during cryopreservation , ice crystals can develop within the cytoplasm of a cell and damage the cellular architecture or lyse the plasma membrane . studies performed on animal models as well as in humans have shown that exposure to low temperatures [ 6063 ] and to cpas   results in depolymerization of the microtubules , sometimes with attendant dispersal of chromosomes . previous studies have shown that changes in culture conditions can alter the organization or structure of mitochondria in the oocytes or embryos of mice . freezing can also denature or damage critical cellular organelles and enzymes , thereby compromising the ability of the embryo to repair structural damage and continue development . actin gives mechanical strength to the cell and plays a major role during cytokinesis , anchoring the centrosomes during mitosis and generating the cell shape . ( 2008 ) , slow freezing and thawing of camel embryos resulted in a modest level of cell death but more widespread disruption of the actin cytoskeleton . their findings agree with our results , which showed that slow freezing cause more disruption of actin structures of embryos as compared to controls and vitrified embryos . the nucleus coordinates all cellular activities , contains the genetic material , and is crucial in the production of proteins . the present results show that the reduction of nucleus intensities is seen in vitrification and slow freezing . as the number of the cells increased , slow - frozen embryos showed the lowest intensity as compared to the normal and vitrified embryos . in this case , most of the chromatin structure may be disrupted in the embryos cryopreserved by the slow freezing method . any disruption that occurs in chromatin distributions will then affect the formation of actin and tubulin and may be blocked the chromosome migration . in addition , the extracellular space became hypertonic due to the increase in solutes compared with that of the intracellular spaces , resulting in water leaving the cell and subsequent cell shrinkage . during warming , a rapid influx of water into the cell occurred due to the increased concentration of solutes within the cell , before the permeating cryoprotectant can be removed . the entry of water is faster than the exit of solutes , thus the cell volume is increased beyond its lytic volume . contraction of the cell membranes during cryopreservation caused a rearrangement of the cell organelles , including the cytoskeleton and the meiotic spindles , which may be a contributing factor to the decrease of tubulin intensities in the vitrified and slow - frozen embryos . based on confocal studies , the slow freezing method showed worse cryopreservation effects compared to vitrification in terms of cytoskeletal damage , which strongly suggests that vitrification is the better procedure for cryopreservation . our study also found that cryopreservation of 2-cell - stage embryos causes the least cytoskeletal injury as compared with 4- and 8-cell stages .
backgroundthis study aimed to investigate the effects of vitrification and slow freezing on actin , tubulin , and nuclei of in vivo preimplantation murine embryos at various developmental stages using a confocal laser scanning microscope ( clsm).material / methodsfifty female mice , aged 46 weeks , were used in this study . 
 animals were superovulated , cohabitated overnight , and sacrificed . 
 fallopian tubes were excised and flushed . 
 embryos at the 2-cell stage were collected and cultured to obtain 4- and 8-cell stages before being cryopreserved using vitrification and slow freezing . 
 fixed embryos were stained with fluorescence - labelled antibodies against actin and tubulin , as well as dapi for staining the nucleus . 
 labelled embryos were scanned using clsm and images were analyzed with q - win software v3.resultsthe fluorescence intensity of both vitrified and slow - frozen embryos was significantly lower for tubulin , actin , and nucleus as compared to non - cryopreserved embryos ( p<0.001 ) . 
 intensities of tubulin , actin , and nucleus in each stage were also decreased in vitrified and slow - frozen groups as compared to non - cryopreserved embryos.conclusionscryopreservation of mouse embryos by slow freezing had a more detrimental effect on the actin , tubulin , and nucleus structure of the embryos compared to vitrification . 
 vitrification is therefore superior to slow freezing in terms of embryonic cryotolerance .