Barex Biochemical Products
The art of semen preservation
The Vitrification Project.
Progress Report 1. The Straw Flooding device and method.
Introduction.
Cryopreservation of sperm cells as well as other biological material has been widely used in reproductive technologies and has resulted in millions of live births. Two principal approaches have been used: conventional (slow) freezing and vitrification.
Slow freezing is the traditional technique and is widely being used with success, whereas vitrification may become a faster alternative method of cryopreservation since vitrification can be done with simple equipment and little training of personnel, as opposed to slow freezing which requires a large investment in equipment and extensive training of personnel.
Further, with the slow freezing technique, one faces the occurrence of damage of cryopreserved biological material due to extra- or intra-cellular ice formation. This damage can be prevented by and large by the use of permeable cryoprotectants such as glycerol and formamides, but these substances are toxic to many cells. Further, in animal sperm cell cryopreservation, egg yolk is used as a non-permeable protector, which introduces a risk of spreading diseases.
Several publications, including meta-analyses, mentioned that vitrification of human sperm was optimal for sperm cryopreservation as vitrification (as opposed to slow freezing) resulted in better motility recovery and higher mitochondrial activity. Increasingly more studies have reported that vitrification, free from any permeable cryoprotectants, performs better than conventional slow freezing for human sperm cryopreservation.
Despite the advantages of vitrification over slow freezing, sperm vitrification has the major limitation of the small volume load that needs to be used to achieve a high cooling rate. Larger volumes would suffer too much from the so-called Leidenfrost effect, which means that nitrogen gas formation acts as a thermal insulator and prevents the desired rapid decrease in temperature.
Several devices and sperm carriers have been developed to better control the volume and speed of vitrification, including the superfine open pulled straw (SOPS), the straw in straw method, CryoLoop™, CryoTip™, and Cryo Bio™’s High-Security Vitrification System (HSV). The devices used to immerse cells in liquid nitrogen differ in that vitrification occurs in either an “open” or “closed” manner. In open systems, the cells come into direct contact with liquid nitrogen, thus allowing rapid cooling rates to occur, whereas in closed systems the cells are completely sealed within a device before plunging into liquid nitrogen. So far these developments have not yet resulted in widespread routine sperm cell cryopreservation (for a review see: https://doi.org/10.1186/s12958-020-00580-5).
Below, the Straw Flooding (SF) method is presented as a new method to vitrify sperm cells and other biological material without the limitation of a small volume load and yet ultra-rapid vitrification by preventing gas formation to act as thermal insulation of the straw during the vitrification process.
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Results.
To prevent nitrogen gas formation that acts as thermal insulator, a method was developed based on the high-speed flow of liquid nitrogen right past a French straw.
The process of vitrification using the so-called Straw Flooding (SF) method is shown in figures 1 and 2 below. The vitrification is started by pulling down the handle (4) of the SF device, whereby liquid nitrogen (11) flows with high speed out of the liquid nitrogen reservoir (2) into the straw carrier (1). The high pressure of the nitrogen gas in the reservoir pushed the liquid with high speed through the carrier. Liquid nitrogen travels at high speed right past the straw. Any gas formation is immediately removed in the high-speed liquid nitrogen flow as shown in figure 2.
The vitrification process takes less than a second. After letting go the handle after 2-3 seconds, the carrier can be removed and the vitrified straw can be slided into a goblet and canister for storage.
Figure 1. Graphical design of the SF device.
Figure 2. Working principle of the SF device.
The longitudinal section of the straw carrier with straw loaded shows the French straw (8) with sperm cells (S). Any nitrogen gas (G) formed during the exchange of thermal energy between the liquid nitrogen and the straw will immediately be removed by the high-speed flow of liquid nitrogen.
The videos below show how the SF device works. Video 1 shows the flow of liquid nitrogen that squirts out of the SF reservoir. Video 2 shows the vitrification process using the SF device. The straw carrier contains the sealed straw with sperm cells diluted in vitrification medium. The carrier is pushed against the adapter and therafter the handle is pushed for 2-3 seconds. Just a few seconds is more than enough to vitrify the diluted sperm without any Leidenfrost effect due to the working principle as outlined in figures 1 and 2.
Video 1. Illustration of flow of liquid nitrogen.
Video 2. Illustration of flow of liquid nitrogen.
To confirm the rapid vitrification process, experiments were carried out to measure the change in temperature in the straw during the vitrification process. For that purpose, a temperature sensor (Tc-T) was brought in the straw and connected to a Graphtek 240 data logger which has a recording speed of 0.1second.
As a reference, the plunging of straws in liquid nitrogen was carried out to determine the change in temperature (with the occurrence of the Leidenfrost effect).
Figure 4 is an image composite of the display of the Graphtek data logger, which shows that upon plunging of the straw in liquid nitrogen, the decrease in temperature of the vitrification medium from room temperature till -196 takes about 18 seconds.
Figure 4. Image of the HGraphtek display showing the temperature in mini straw (red line) and a medium straw (blue line) after plunging of the straws in liquid nitrogen.
Figure 5 shows the rate of temperature change in the straw when vitrification is done using the SF device. Figure 6 shows the rate of temperature when only the thermocouple is present in the carrier. The display show then the response time of the thermocouple. These data show that the change in temperature of the vitrification fluid in the straw from room temperature to -196 °C is realized in a split second.
Figure 5. Image of the Graphtek display showing the rate of temperature change of the thermocouple when present in a straw subjected to a flow of liquid nitrogen in the SF carrier.
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Figure 6. Image of the Graphtek 240 display showing the rate of temperature change of the thermocouple when subjected to a flow of liquid nitrogen in the SF carrier.
Conclusions.
The work of Prof. Manuel Hidalgo and his coworkers (University of Cordoba, Spain) made clear that vitrification of stallion semen in a straw using the double straw method is an efficient and valuable vitrification method to cryopreserve stallion semen. They have published their vitrification methods in a series of papers (for a review see "Recent advances in donkey sperm vitrification" : https://doi.org/10.1111/rda.13995 ).
The current research aimed to improve their vitrification method to make the routine application on a large scale possible. The results that are presented here show that using the SF method, it is very well possible to efficiently and effectively vitrify stallion semen on a large scale in French straws.
It was shown that the vitrification process using the SF device takes only a split of a second, while plunging a straw in liquid nitrogen takes around 20 seconds. In our unreported experiments, it was shown that in the case of the double straw method the vitrification process takes between 30 and 40 seconds.
The SF device is simple, inexpensive, efficient, and effective, and has therefore the potential to become a valuable tool towards a universal vitrification protocol for semen as well as other biological material.