Effect of Cryoprotective Solutions on Survival and Teratogenicity of Prussian Carp Embryos ( Carassius auratus gibelio Bloch , 1783 )

There was studied the effect of cryoprotective solutions, belonging to different classes of chemicals, on the survival and abnormal development of Prussian carp (Carassius auratus gibelio Bloch, 1783) embryos at the stage of development corresponding to 6 somites. The embryos were incubated during 60 minutes in 10% solutions of cryoprotectants dimethyl sulfoxide (DMSO), 1,2-propane diol (1,2-PD), 1,3-propane diol (1,3 PD), 1,3-butane diol (1,3-DB), 1,4-butane diol (1,4-BD), 1,2-metaoxyethane (1,2-MOE), glycerol, ethylene glycol (EG), methanol, polyethyleneoxide (PEO-1500) and sucrose. Thereafter, the embryos were washed and traced to the stage of development of free-swimming larvae. The highest survival rates for the embryos of Prussian carp (54–67%) was observed in 10% solutions of 1,2-PD, 1,3-PD, EG, PEO-1500, and sucrose, providing the preservation of normally developing embryos close to the control level. Cryoprotective solutions of 1,3-BD, 1,4-BD had higher toxicity: the survival of embryos in those made 40–49%. In the solutions of 1,2-MOE, DMSO, glycerol and methanol cryoprotective agents the survival rate of embryos was minimal. The highest level of teratogenic embryos was observed after incubation in the cryoprotectant solutions of 1,2-MOE and methanol.

To date there are effective cryopreservation methods developed only for fish sperm [25] and not for the embryos [4].The results of the research performed in the embryos of various fishes and at different developmental stages in particular confirm the fact that the tested cryoprotective agents constituting the cryoprotective media and consequently the whole cryopreservation methods are not efficient.It is known that the preparing of a biological specimen for a deep cooling includes the incubation in cryoprotective media containing penetrating and/or non-penetrating cryoprotective components.The majority of embryos dies in cryopreservation medium already during incubation mainly due to osmotic factor [21] and toxic effect of cryoprotectants rendered to embryos [2].Thereby the need in new approaches to solve this task appears.Some authors hypothesized the necessity to use multicomponent cryoprotective media with a high concentration of cryoprotectants and to enhance the cooling rates up to ultra-rapid ones to facilitate vitrification [6,19,20].Development of effective cryopreservation methods is tightly related to the investigations of the effect of cryoprotectants on survival of embryos which could be used to obtain cryoprotective media.Herewith it is primarily necessary to study the transport of water and cryoprotective solutions into fish embryos [3].
Heterogeneity of membrane complexes of chorion, blastoderm and yolk syncytium is the main obstacle for effective dehydration and saturation of fish embryo cells with cryoprotective solution [11,13,27].Herewith not only the survival rate of incubated in cryoprotective media embryos alters but also does the level of their teratogenicity.Rise in the index of embryo survival post incubation in cryoprotective media could be achieved by reducing a cytotoxic effect of some cryoprotective components of the medium [8].In this connection the study of the effect of cryoprotective solutions on morphofunctional integrity of fish embryos would allow to select the substances with the lowest negative effect, which could thereafter be the potential components of cryoprotective media [7].Traditionally the studies concerning fish embryo resistance during preparing to a deep cooling are performed with such cryoprotectants as dimethyl sulfoxide, methanol, glycerol and ethylene glycol [8,10,18,22].However, the experimental data obtained by various research teams are segmentary and require a systematization and complementation.
The research aim was to compare study the effect of cryoprotectants belonging to different classes of chemicals on morphofunctional preservation of Prussian carp embryos.
Растворы 1,2-МОЭ, метанола, глицерина и ДМСО в концентрации 10% снижают уровень выживаемости эмбрионов карася при инкубировании of 6 somites.Fish were caught during spring-summer period from their natural habitats.The preference was given to mature males aged 3-4 years.The procedure of gonadotropic injection to females and males, as well as derivation of reproductive products and insemination were performed in accordance with fish breeding recommendations [9].The embryos were incubated at room temperature (18...21°C) in Petri dishes by 100-150 in each.
Prussian carp embryos at developmental stage corresponding to 6 somites were incubated with cryoprotectants solutions for 60 min.To remove cryoprotectants the embryos were thrice transferred to water with parameters optimal for incubation [24].State of embryos and teratogenic rate at all the stages were visually estimated in accordance to the maps of embryonic development [24] using microscope MBS-9.The embryos in the studied groups and control were monitored to the development stage of free-swimming larva.
The obtained results were analyzed using the Statistica software (Statsoft, USA).The differences between the groups were considered as statistically significant at p < 0.05.

Results and discussion
Fig. 1 demonstrates the indices of survival of Prussian carp embryos after incubation in the studied cryoprotective solutions.
Successful cryopreservation of biological specimen especially when using the vitrification method mainly depends on effective saturation of cells with cryoprotectants.M. Hagedorn et al. [13] studied the ability of DMSO, 1,2-PD and methanol to saturate the zebra fish (Cyprinidae) embryos.Method of magnetic resonance allowed to show that only methanol penetrated to the embryo during 15 min and the other cryoprotectants needed much longer time period.The results explain our findings on the effect of methanol in terms of the survival rate as well as an increase of teratogenically developing embryos of Prussian carp as concerned with long incubation time in the methanol solution.
Veprintsev B.N. et al. [23] investigated the survival of loach (Misgurnus fossilis) embryos after 60-min exposure in 10% DMSO solution at the developmental stage corresponding to the start of organogenesis (formation of 3-13 somites).A high preservation rate of loach embryos (98%) was explained by the authors by a low permeability of cell membranes for the substances of a high molecular weight as observed in most other fresh water fishes.Some investigators also point to a low or moderate toxicity of DMSO solution in respect to fish embryos, enabling to use it in cryoprotective media [5,6,22].However, these experiments were performed in the embryos of marine species (Sciaenops ocellatus, Pargus major and Scophthalmus maximus), known to be more resistant to the effect of cryoprotectants [4].
It has been demonstrated that the rise in concentration of cryoprotectants affect significantly the survival of embryos [7,8].Therefore we considered as expedient to trace the development of Prussian carp embryos up to the stage of free-swimming larva, since a cytotoxic effect may affect the development of embryos later.Data on the effect of the studied cryoprotectants on abnormal development of Prussian carp embryos are presented in Fig. 2.
It is seen that maximal level of teratogenic embryos was observed after incubation in the solutions of cryoprotectants 1,2-MOE, DMSO, glycerol and methanol.In the externally fed larvae we have found signs of wet perivitelline syncytium, disorders in segmentation of muscular body and asymmetry of cranial compartment.No statistically significant differences of the studied indices were observed in the solutions of diols, DMSO and glycerol.
Previously it has been shown that teratogenic embryos development depends directly on the ability of cryoprotectant to penetrate via plasma membranes and is characterized with more distant consequences, that is specific for each fish species [17,19].
established that for a complete removal of cryoprotectants from all structural elements of an embryo the participation of additional ingredients contributing to washing (e. g. pronase) is necessary.Just in this case, the embryos including those being teratogenically developed kept a high survival rate.
In the result of our experiments it has been established that 10% cryoprotective solutions 1,2-PD, 1,3-PD, 1,3-BD, 1,4-BD, glycerol, EG, PEO-1500 and sucrose provided following 60-min incubation a viability of normally developing embryos close to the control.The effect of these solutions in terms of teratogenicity of developing embryos had statistically significant differences vs. the control.So, there is a need of developing new methods to remove the cryoprotectants out of embryos.
There is an opinion that creation of the cryoprotective media based on either DMSO or methanol needs the reduction of their concentration and decreasing a total incubation time, and in its turn this requires further studies [12].

Conclusions
1. Solutions of cryoprotectants 1,2-PD, 1,3-PD, 1,3-BD, 1,4-BD, EG, PEO-1500 and sucrose in 10% concentration provide quite a high survival rate of Prussian carp embryos following 60 min incubation if compared with the control and could be recommended to be used as the components of multicomponent cryoprotective media.