Low-temperature preservation of sterlet reproductive cells (Acipenser ruthenus Linnaeus, 1758) by the acoustic-mechanical influence method

. To preserve reproductive cells of rare and endangered species of sturgeon fish using low-temperature cryopreservation methods, acoustic-mechanical impact with the use of piezoactuators was used. As a result of the research, the optimum parameters of signal frequency and time of impact on sturgeon cells were established, at which a high survival rate of sperm after defrosting was observed.Using mathematical modelling, the mechanism of acoustic-mechanical influence was studied, which consists in the wave and mechanical action created by the piezoactuator, which leads to acceleration of the process of diffusion of the cryoprotector inside the cells.For the first time a new methodological approach to low-temperature preservation of fish reproductive cells with the use of acoustic-mechanical impact has been proposed, which makes it possible to preserve genetic material with high survival rate and quality indicators.


Introduction
In order to improve the quality of cryopreserved reproductive cells, the cells are affected by changing factors of different nature (mechanical, chemical, physical) in the process of lowtemperature preservation.As a result, the penetration of protectors inside the cells is improved, their protective effect is increased and, ultimately, the percentage of sperm survival is increased [1][2][3].
In livestock breeding, artificial insemination of farm animals using defrosted semen has an industrial basis, in contrast to fish farming (aquaculture), and research in this area is mainly aimed at intensification of techniques.However, the previously established standard of motile defrosted sperm, which does not exceed 45-50 %, is still used in practice.An increase in the number of surviving sperm after deconservation is possible not only through the use of stabilising additives, but also through the influence of acoustic or electromagnetic factors [4][5][6].
The application of a new scientific approach to methods of long-term preservation at low temperatures of genetic and reproductive material of hydrobionts using acoustic-mechanical effects opens up great opportunities for the creation of new cost-effective biotechnologies that will make it possible to make the transition to aquaculture farms of a new type, increase the stability of aquaculture as a whole, and make conservation measures to save rare and endangered species more effective [7][8][9].
To improve the quality of defrosted fish semen, various kinds of stimulating effects are used, such as electric and magnetic fields, acoustic effects, ultrasound, etc.However, acoustic-mechanical influence has not been studied on reproductive cells of sturgeon fish, which is a new and actual direction in the field of low-temperature preservation [10][11][12].
The use of new methods will contribute to increasing the fertilising ability of fish spermatozoa after long-term low-temperature storage at -196 ºC liquid nitrogen temperature and further improve the quality of offspring obtained from cryopreserved sperm.
The aim of the research is to determine the efficiency of accoustic-mechanical action using piezoactuates during deep freezing of sterlet reproductive cells (sperm).

Material and research methods
To influence the cells during the period of interaction with the cryoprotectant, an experimental setup was created, which is designed to excite acoustic waves in liquids, consisting of the following elements: a test tube with a piezoelectric transducer glued on it, a Tektronix AFG 3022B functional signal generator, a signal amplifier based on piezo drivers and a LeCroy Wavesurfer 422 oscilloscope.A sine wave with a given frequency and amplitude was generated using the function generator.This signal was then fed to an amplifier and then to a piezo transducer.The transducer was a disc made of piezoceramic material, which due to the piezoelectric effect allows to convert the electrical signal into mechanical oscillations.The latter, in turn, are transmitted to the test tube and the liquid (cryosolution and cellular material) inside.An oscilloscope was connected between the converter and the amplifier to monitor the output characteristics of the amplified signal.
After exposure, semen samples were poured into plastic Ependorf tubes and sent for equilibration in a refrigerated chamber for 40 min.After the time had elapsed, semen quality was assessed.
Samples were frozen using a Planer freezer Kryo 560-16, which performs programmable, controlled, logged and reproducible batch cooling of the object to a preset temperature.Dewar transport vessels were used to transport the reproductive material.After bringing the cryopreserved material to the storage temperature (usually (-196)ºC -the temperature of liquid nitrogen), the object is placed for a long period of time in a large-capacity cryopreservatory.In our experiments, frozen semen samples were withdrawn after 1-3 days and 7 days.
The samples were frozen in steps: 3 °C/min for 4 minutes, then 10 °C/min for 10 minutes.After the temperature of samples reached -70 °C, they were placed in Dewar vessels with liquid nitrogen for storage.
In the first series of experiments, the exposures were carried out at the following parameters: 20 Hz (A), 500 Hz (B), 5.8 kHz (C), at a voltage of 10-15 V, and the exposure time was 1 minute.The parameters were chosen on the basis of preliminary studies and analysis of modern scientific literature.
In the second series of experiments, low-temperature preservation of sterlet sperm was carried out according to the method developed earlier [3,4].To determine the exposure time in this series of experiments, exposure for 1 min, 1.5 min with parameters A -400 Hz (A); B -500 (B) Hz; C -1 kHz (C) was carried out.
After storage of samples in liquid nitrogen for three days, defrosting was carried out and the quality of reproductive cells was determined.

Results and discussion
In the first series of experiments, semen samples №1, №3, №5, №6 with different reproductive qualities were selected for research (Table 1).This was done in order to determine whether acoustic-mechanical effects would help to improve the reproductive qualities of sperm with low native parameters.The results of the semen quality study after defrosting are shown in Figure 1.Since only spermatozoa with active translational movement have fertilising capacity, the oscillatory movement of sperm was not taken into account in the results.As can be seen from Figure 2, the duration of exposure plays an important role in acousticmechanical treatment of sterlet sperm.Thus, when exposed for 1 min, there is an increase in the survival rate and percentage of actively moving spermatozoa after thawing, while at the duration of 1.5 min.-On the contrary, there is a decrease in sperm activity compared to the control.Theoretical substantiation of the mechanism of acoustic-mechanical influence on sturgeon sperm during cryopreservation served as a platform for the experimental part of the research.Using mathematical modelling, it was possible to study the mechanism of acousticmechanical influence, which consists in the wave and mechanical action created by the piezoactuator, which leads to acceleration of the process of diffusion of the cryoprotectant inside the cells.When testing piezoactuators in the process of cryopreservation, it was found that acousticmechanical action with a signal frequency of 500 Hz for 1 minute had a positive effect on the activity of defrosted sperm.
Duration of progressive movement of spermatozoa is an important fish breeding indicator, because only actively moving sperm are able to fertilise fish eggs.According to fish breeding indicators, good quality semen is considered to be that in which the sperm are actively moving for at least 5 minutes.As can be seen from Figure 4, the best indices are observed in the variant with duration of acoustic-mechanical influence for 1 min.
The best activity indices are observed in variant B (cells of male #6), compared to native sperm.This indicates that acoustic-mechanical impact with a signal frequency of 500 Hz has the best effect on reproductive qualities of defrosted semen.The semen of males #1 and #3 showed zero results for all treatments, the semen of male #5 showed low results, but the results were higher than the native semen at 500 Hz signal frequency.This implies that acoustic-mechanical exposure improves sperm quality compared to native sperm during cryo-freezing, whereas the performance of native sperm before freezing was higher.The results of the first experiment were preliminary and required further investigation.
In the second series of experiments, when the frequency exposure range was between 400 Hz and 1kHz, positive results were obtained in two variants of the experiment, the best results were obtained on the sperm of male #6 when exposed to 400 and 500 Hz, the percentage of surviving cells was 60 and 55, respectively, compared to the control, where the survival rate was 40%.The sperm of male #5 also showed positive results compared to the control when exposed to 400 and 500 Hz, with cell survival rates of 30% and 20%, respectively.The rates were lower in the control and amounted to 10%.The results of exposure to sperm from male #8 were low compared to the control, but this again indicates that high quality sperm should be used for cryopreservation.In male #8 the indices were low, sperm score on Persov scale was 3-4 points, activity up to 25%.
The results of the experiment according to the time of exposure showed that when exposed for 1 minute the activity of reproductive cells was higher and was 30.7 % in variant A, 23.3 % in variant B, when the time of exposure was increased up to 1.5 minutes the indicators were lower.
Thus, in the technology of low-temperature preservation of fish reproductive cells at the stage of equilibration it seems reasonable to use piezoactuators at the first bending mode, to recommend the impact frequency of 400-500 Hz at exposure time of 1 min.

Table 1 .
Quality indicators of fresh sterlet semen