Introduction
The animal reproductive industry has been using liquid nitrogen (LN2) tanks to freeze and store frozen semen for several decades. More recently with the rapid development of embryo transfer technology in different species, it is being used for storing frozen embryos. Because LN2 tanks are a tool that farmers, artificial insemination technicians, embryo transfer practitioners, veterinarians, animal reproductive scientists, semen banks and researchers use everyday, this paper has been written to provide a basic knowledge and understanding of this amazing product and how to use it carefully and more efficiently.
Cryogenics Liquids
Cryogenic liquids or "Cryogens" are liquefied gases that have been cooled below ambient temperature, pressurized, and kept in their liquid state at very low temperatures. Cryogenic liquids have boiling points below -150°C (-238°F).
Examples of cryogens are the inert gases such as nitrogen, helium, neon, argon and krypton. Additional examples are the flammable gases such as hydrogen, methane, natural gas and oxygen.
Cryogens at room temperature and normal pressure are found as gases. In order to transform these gases into cryogenic liquid, they must be cooled below room temperature and then pressurized to liquefy them.
All the cryogens have three important characteristics:
· They are extremely cold. The vapors and gases released from these cryogens are also extremely cold.
· Very small amounts of these liquid cryogens can expand into very large volumes of gas that can displace air. For instance, 1 liter of liquid LN2 can form 695 - 700 liters of nitrogen gas when warmed to room temperature (20 - 21°C).
· According to WHMIS, cryogenic liquids are "compressed gases". Cryogens will create high gas pressures inside the storage containers, and therefore they require a relief vent/valve to allow the gases to escape from the container.
Nitrogen (N)
Nitrogen is a chemical element with the symbol N. Its atomic number is 7 in the periodic table. Nitrogen constitutes 78.03% by volume and 75.5% by weight of the Earth’s atmosphere making it the largest single component of the air. It is colorless, odorless, and tasteless. It is an important component of all living tissues and amino acids.
Nitrogen is classified as an "inert" gas due to its non-reactive nature with many materials. Nevertheless, nitrogen can form certain compounds under the influence of chemicals, catalysts, or high temperature. For example, when nitrogen is combined with hydrogen in the presence of a catalyst, ammonia is formed.
Liquid Nitrogen (LN
2
)
Liquid nitrogen (LN2), the cryogenic fluid used in the reproductive industry, is inert, colorless, odorless, non-corrosive, non-flammable, and extremely cold. It is produced industrially in large quantities by fractional distillation of liquid air.
When appropriately insulated from ambient heat, LN2 stays at -196°C or -320.5°F, which is the nitrogen boiling point. Because it maintains temperatures far below the water freezing point, LN2 is very useful in a wide range of applications as an open-cycle refrigerant and cryoprotectant including:
Immersion freezing and transportation of food products.
Cryopreservation of blood, reproductive cells (sperm, oocyte, embryos), and other biological materials.
Cryonic preservation of humans and pets in the unproven hope of future reanimation.
Study of cryogenics per se.
Demonstrations for science education.
Coolant for highly sensitive sensors and low-noise amplifiers.
Dermatology for cryotherapy removal of potential malignant skin lesions.
Coolant supplement for overclocking central processing units, graphics processing units, or computer hardware.
Coolant during machining of high strength components.
Working fluid in a binary engine.
Precaution when working with Nitrogen and LN
2
Rapid release of nitrogen gas into an enclosed space can displace oxygen in the air to levels below that required to support life, representing an asphyxiation hazard. The human carotid body, located near the bifurcation of the carotid artery, is an anatomical organ that measures changes in blood pressure and the composition of arterial blood flowing past it, including the partial pressures of oxygen and carbon dioxide. It is also sensitive to changes in pH and temperature. Unfortunately, when somebody aspirates air with higher concentration of nitrogen gas than normal, the carotid body works relatively slow and this low-oxygen concentration sensing system (hypoxia) will not react in an efficient way to prevent potential asphyxiation. When inhalation of nitrogen is excessive, symptoms such as dizziness, nausea, vomiting, loss of consciousness and death can happen. Death may happen from errors in judgment, confusion, or loss of consciousness that prevents self-rescue. At low oxygen and high nitrogen concentrations, unconsciousness and death may occur in seconds and without any warning. Because of this asphyxiation hazard, when working with LN2 in a confined or enclosed space it is recommended to monitor oxygen concentration with specialized ambient oxygen monitor-alarms that are available in the market (Fig. 1).
Figure 1. Example of an oxygen monitor to measure ambient oxygen concentration in confined rooms. This type of oxygen monitor activates when the oxygen concentration reaches below the set point, which is normally 19% or 19.5% O2. (Picture obtained from Alpha Omega Instruments website).
LN2 is capable of causing instant frostbite on contact with living tissue. Direct skin contact with LN2 causes severe frostbite (cryogenic burns) within moments to seconds depending on the form of liquid nitrogen (liquid vs. mist) and surface area of the nitrogen-soaked material. It is important to know that soaked clothing or cotton can cause more rapid skin damage than a spill of direct LN2 to the skin, which for a few seconds is protected by the Leidenfrost effect. The Leidenfrost effect is a phenomenon in which a liquid, in near contact with a mass significantly hotter than its boiling point, produces an insulating vapor layer which keeps that liquid from boiling rapidly. This effect happens when somebody dips a wet finger in molten lead or blowing out a mouthful of liquid nitrogen, both situations causing no injuries to the demonstrator.
The best practice to prevent frostbite when handling LN2 is to wear personal protective equipment such as a full-face shield over safety glasses, loose–fitting thermal insulated or leather gloves, long sleeve shirts, and trousers without cuffs.
Storage of Cryogenic Liquids
Cryogens are shipped and stored in thermally insulated containers. The cryogenic containers are particularly designed to resist rapid temperature changes and extreme temperature difference. There are two types of tanks commonly used in the reproductive industry:
Liquid Dewar flasks: They are non-pressurized, vacuum –jacketed vessels similar to a "thermos bottle". They come with a loose fitting cap or plug that helps to prevent air or moisture entering and also allows excess pressure to vent.
Liquid cylinders: They are pressurized containers specifically designed for cryogenic liquids. This type of container has valves for filling and dispensing the cryogenic liquid, and a pressure-control valve with a frangible (bursting) disk as backup protection. Three major types of liquid cylinders have been designed for dispensing either liquid or gas, gas only or liquid only.
Tanks for Shipping or Storage of Semen / Embryos
Dry Shippers
Dry shippers (Fig. 2) are Dewar flask containers designed for transportation of frozen biological specimens (semen or embryos) in LN2 vapor at temperatures around -190°C. They are made of lightweight aluminum and work by absorbing LN2 into a thick layer of hydro-phobic material that surrounds the inner cavity of the container where the semen is stored. The dry shippers must be properly charged by filling with LN2 to the point of saturation of the absorbing material and then removing the excess LN2 to provide maximum holding time. The long holding time of dry shippers is obtained because there is superior insulation created by a double-wall aluminum shell that is filled with foil and also vacuum sealed (Fig. 3). A unique lid plugs the dry shipper’s neck tube, helping to create the insulated interior environment of LN2 vapor.
Special cases or cartons are available from the manufacturers to protect the dry shippers from potential damage in transportation and to prevent the LN2 from spilling by ensuring the system remains upright at all times (Fig. 4).
Figure 2. Dry shipper container commonly used for transport of frozen semen or embryos.
Figure 3. Interior schematic view of a dry shipper. A - Aluminum wall. B - Locking tab. C – Neck tube. D – Vacuum chamber. E – Hydrophobic absorbent material shown in blue. F – Insulation shown in yellow. (Picture obtained from the MVE Bio-Medical Vapor Shippers catalogue, Chart Industries, Inc - See reference list below).
Figure 4. Dry shipper protective case.
Dry shippers can be classified according to:
It is the amount of time that the dry shipper will be capable of storing the specimen in LN2 vapor at the required temperature (-190°C) during transportation. The static holding time is measured in days and helps to calculate the shipping time of specimens. In the marketplace, there are dry shippers with static holding times that vary between 8 and 24 days. The static holding time depends on the LN2 evaporation rate of the shipper, which is calculated in litters evaporated per day (liters/day). At the same time, the evaporation rate depends on the dry shipper’s LN2 capacity measured in liters and the size of the neck opening. The liquid nitrogen capacity for dry shippers varies between the different models available; this usually ranges between 1.5 to 7 liters of LN2.
Storage Capacity
This is a measure of the maximum amount of straws that can be stored in a dry shipper for transportation. Usually dry shippers have only one canister, which will vary in diameter and height depending on the model. Therefore, the storage capacity will depend on the size of the canister, the type of straw being used (0.5 or 0.25 ml straws), and whether or not the straws are packaged on aluminum canes.
Storage Tanks
Biological specimens such as frozen semen and embryos are normally packaged in straws and stored in LN2 tanks (Fig. 5) with similar characteristics to a liquid Dewar flask described previously. Storage tanks are large, metal, vacuum-sealed liquid nitrogen refrigerators encased within an extremely efficient insulation system. They are made of aluminum and have two separate chambers: an inner and an outer chamber (Fig. 6). The space between the two chambers is filled with foil and special paper, and the air is removed to create a partial vacuum in this area. The vacuum increases the insulating effect and is the major effective property of the tank. A unique lid is used to plug the neck tube and create further insulation for the liquid nitrogen in the interior of the tank.
Figure 5. Left: Picture of a semen/embryo storage tank with a LN2 capacity of 22 liters and holding time of 40 days.
Right: Top view of the same storage tank after removing the lid. This tank has six canisters that are enumerated individually and can be seen in the interior of the tank.
Figure 6. Left: Interior schematic view of a 6 canister semen/embryo storage tank with the inner chamber with LN2shown in blue. A – Protective cover lid. B – Top. C – Aluminum wall. D – Neck tube. E – Locking tab. F – Lid or plug. G- Vacuum chamber. H – Bottom spider design for individual allocation of canisters. J – Insulation shown in yellow.
Right: Semen storage tank with a measuring stick for LN2. (Left picture obtained from the MVE SC Series - Aluminum Storage Tanks catalogue, Chart Industries, Inc).
As long as any LN2 is present inside the tank, LN2 storage tanks can keep semen and embryos at -196°C. The ideal scenario is to have the LN2 tank filled between half and full capacity. The recommended minimum volume of LN2 present in the tank is at least 5 cm (2 inches) in order to prevent any damage to the stored specimens. Ideally, the LN2 level should be monitored every week with a calibrated plastic measuring stick (Fig. 6).
The inner chamber is the area that stores the LN2 and contains the frozen specimens, and is actually held from the outer shell by the neck tube (Fig. 6). Storage tanks are very delicate. Any strong or excessive swinging motion could cause rupture of the neck tube creating vacuum loss and tank failure.
Storage tanks can be classified according to:
Static Holding Time
This is the amount of time that the tank will be capable of storing the specimen in LN2 at -196°C. The static holding time for storage tanks is measured in days and is used to calculate how often the tank needs to be refilled with LN2 and their estimated working time. The static holding time for these tanks varies considerably, and it may be as low as 17 days or as long as 340 days. The static holding time depends on the LN2 evaporation rate of the tank, which is calculated in liters evaporated per day (liters/day). The evaporation rate depends on the tank’s LN2 capacity measured in liters and the size of the neck opening. The liquid nitrogen capacity also varies between the different storage tank models available in the market place. In general, the LN2 capacity for the storage tanks used on farms, in veterinary clinics or by artificial insemination, technicians can range between 3.6 to 50 liters depending on the model and the manufacturer (Fig. 5). In the bovine artificial insemination industry, companies that collect, process, store and distribute bull semen require larger storage tanks with LN2 capacities in the hundreds or thousand of liters (Fig. 7). These tanks can also be used in frozen semen banks.
Figure 7. Top: Picture of liquid nitrogen tanks for storage of high volume of frozen semen/embryos. The left tank has a LN2 capacity of 370 liters, the middle one 756 liters, and the right one 1,672 liters.
Bottom: Interior schematic view of a high volume storage tank for semen or embryos. A – Offset neck design. B – Aluminum lid in yellow. C – Rotating interior tray. D – Stainless steel wall. E – Annular filling lines. F – Casters. G- Rack stand. H – Step-up platform. (Pictures obtained from the MVE Eterne Series – 190°C Vapor Storage Catalogue, Chart Industries, Inc).
Storage Capacity
This is a measure of the maximum straw holding capacity of the storage tank. Storage tanks normally have several canisters (6, 9 or 10 canisters) (Fig. 5) that vary in diameter and height depending on the tank model. The tank’s storage capacity will depend on the size of these canisters, the type of straw being stored (0.5 or 0.25 ml straws), and whether or not the straws are packaged on aluminum canes. As a rule of thumb, the manufacturers calculate the maximum storage capacity of tanks by using only 0.5 ml straws. When using aluminum canes, it is important to remember that five 0.5 ml straws can be stored in a 9.2-mm goblet, and that one aluminum cane can hold two 9.2 mm goblets (top & bottom level). This means that ten 0.5 ml straws can be stored per aluminum cane using 2 goblets (Fig. 8) and that several full aluminum canes are required to fill each canister (Fig. 9). The aluminum cane system is widely used for packaging frozen semen from different animals and it is commonly used in the reproductive industry worldwide. Each aluminum cane has a labeling tab on the top that allows the user to identify the 10 semen straws stored in that cane (Fig. 9).
Figure 8. Left: Aluminum cane and plastic goblets (9.2 mm diameter) with 0.5 ml straws before being assembled.
Right: After assembling the parts. Notice top and bottom location of the goblets in the cane.
Figure 9. Observation of the interior of a semen storage tank. One of the canisters has been retrieved to the center of the tank to show 7 aluminum canes (with 10 mm diameter goblets) located inside the canister and with extra room for storing more cane units.
On the other hand, some companies report maximum storage capacity of a tank as "Bulk storage" in 1 or 2 levels. In this type of storage, a much larger plastic goblet with a diameter very close to the internal diameter of the canister is used to store the straws (Fig. 10). One level bulk means that only one goblet full of straws is used in all the canisters (Fig. 10). Two level bulk means that two goblets full of straws are used in all the canisters, one on top of the other (Fig. 11). Because there is no need for using aluminum canes the storage capacity is increased automatically. With 2-level bulk storage, the tank capacity can be increased by 2 or 3 times, when compared with storage on aluminum canes. Bulk storage is routinely used by bovine artificial insemination companies to store large quantities of frozen semen for long periods of time. To locate and identify bulk stored semen, the visible portion of the canister handle is labeled and the information recorded.
Figure 10. Left (a): View of a 65 mm plastic goblet showing its high storage capacity, around 365 (0.5 ml) straws.
Right (b & c): Interior view of a semen storage tank that has 1-level bulk straw storage. Each canister is 79 mm in diameter and contains only one 65 mm diameter goblet full of 0.5 ml straws located at the bottom of each canister. The total capacity of this tank, using 65 mm goblets in 1 level bulk storage would be approximately 2,190 (0.5 ml) straws.
Figure 11. Interior view of a tank showing 2-level bulk straw storage. The canister is 79 mm in diameter and contains two 65 mm diameter goblets full of 0.5 ml straws, locating one goblet on top of the other inside each canister. The total capacity of this tank, using 65 mm goblets in 2-level bulk storage in 6 canisters would be approximately 4,380 (0.5 ml) straws.
As a rule of thumb, the user can nearly calculate the capacity of a storage tank for 0.25 ml straws by doubling the capacity mentioned for 0.5 ml straws. For instance if a storage tank has a maximum capacity of 720 (0.5 ml) straws, it can be inferred that the tank will have a capacity of approximately 1,400 (0.25 ml) straws.
The following are important procedures to ensure maximum holding time in a LN2 cryogenic storage tank:
LN
2
cryogenic storage tanks should be kept in a cool, dry, clean, and well ventilated room or area.
To protect the aluminum from corrosion, tanks should be placed elevated above concrete or wet floors. Manufacturers have specially designed roller bases with wheels that allow LN
2
tanks to be moved easily.
LN
2
tanks should be located in an area that allows daily visual observation. In case of tank failure (i.e. neck fracture or loss of vacuum), frosting formation on the tank’s neck can be observed and corrective measures must be taken quickly.
The LN
2
tank level should be checked weekly and recorded in a LN
2
tank log for this purpose. This weekly task will help to calculate the evaporation rate for each individual tank, and how often it should be refilled.
It is recommended to top off a LN
2
tank when the measuring stick shows half tank capacity.
Perform an inspection of the lid regularly. When defective or damaged, replace it to improve static holding time.
Goblets
As previously mentioned, frozen semen straws are often packaged in plastic goblets inside the semen storage tanks. Goblets are normally round in shape, although there are other shapes available, such as polygonal, hexagonal, etc. Round goblets are usually classified and differentiated by their diameter in millimeters (mm) (Fig. 12 and Table 1). The larger the diameter of a round goblet, the higher the straw storage capacity will be (Table 1).
Figure 12. Comparison of size and capacity of different plastic goblets used to store semen straws in storage tanks. The goblet containing yellow straws is 10 mm in diameter; the one having red straws is 13 mm in diameter; the one with transparent straws is 35 mm in diameter; and the one with blue straws is 65 mm in diameter.
Table 1. Comparison of Plastic Goblets Straw Capacity
Goblet diameter (mm)
0.5 ml straw capacity (units)
0.25 ml straw capacity (units)
7.1
-
5
9.2
5
10
10
7
14
13
10
25
35
100
225
39
125
275
42
145
325
53
250
550
65
365
820
The smaller goblets (7.1, 9.2, 10 and 13 mm) are normally put on canes for shipping, distribution and storage of semen (Fig. 8, Fig. 9 and Fig. 12). The larger goblets (35, 39, 42, 53 and 65 mm) do not require canes and are placed directly inside the canister (Fig. 10, Fig. 11and Fig. 12). As previously described, larger goblets are used for bulk storage of frozen semen.
Calculating Storage Tank Capacity
The following examples will assist someone buying a semen/embryo storage tank to calculate the storage capacity quickly.
Using canes and small goblets, a tank manufacturer describes a tank with the following characteristics:
No. of 1/2 cc straws (10 per cane): 1,260
Number of canisters: 6
Canister diameter: 56 mm
The user wishes to use 10 mm round goblets, aluminum canes that hold 2 round goblets per cane and is going to store semen packed in either 0.5 ml or 0.25 ml French straws.
A 10 mm goblet can store 7 straws (0.5 ml) or 14 straws (0.25 ml) inside (Table 1). This means that each aluminum cane can store a maximum of 14 (0.5 ml) straws or 28 (0.25 ml) straws. First determine how many aluminum canes can be stored per canister without being jammed. In a 56 mm diameter canister, it is possible to store 15 aluminum canes with 10 mm goblets. Therefore, the capacity per canister will be at least:
15 canes X 14 straws = 210 straws (0.50 ml) per canister
15 canes X 28 straws = 420 straws (0.25 ml) per canister.
The tank has six canisters. This means that the storage capacity of the tank when full is at least:
6 canisters X 210 straws = 1,260 straws (0.50 ml straw)
6 canisters X 420 straws = 2,520 straws (0.25 ml straw)
To store this amount of straws, the user will require 90 aluminum canes and 180 goblets (10 mm diameter).Bulk storage.
The manufacturer describes the tank for bulk storage as:
No. of 1/2 cc straws (1 level bulk): 1,764
Number of canisters: 6
Canister diameter: 56 mm
The user wishes to use 53 mm round goblets that fit inside the 56-diameter canister and is going to store semen packed in either 0.5 ml or 0.25 ml French straws.
A 53 mm goblet can store at least 250 straws (0.5 ml) or 550 straws (0.25 ml) (Table 1).
If using 1-level bulk storage, only 1 goblet (53 mm diameter) would be placed per canister. The maximum capacity of the tank would be at least:
6 goblets X 250 straws (0.5 ml) = 1,500 straws (0.5 ml)
6 goblets X 550 straws (0.25 ml) = 3,300 straws (0.25 ml)
According to these calculations, when using one 53 mm goblet per canister, the 1 level bulk maximum storage capacity of the tank could not be reached. The explanation for this discrepancy may be that a 1 or 2 mm larger goblet (54 or 55 mm goblet) was used in the original capacity calculation allowing for storage of more straws per goblet/canister, or it may be that the capacity for the 53 mm goblet was a conservative estimate and it could have actually stored more straws than we calculated. In general, goblets are usually not packed to maximum capacity for storage purposes.
An alternative for more storage in a tank is to use 2–level bulk storage. Two goblets (53 mm diameter) are stored per canister instead of only one. Then the maximum capacity of the tank would be:
12 goblets X 250 straws (0.5 ml) = 3,000 straws (0.5 ml)
12 goblets X 550 straws (0.25 ml) = 6,600 straws (0.25 ml)
When using 2-level bulk storage, the tank’s capacity for 0.5 ml straws has been almost doubled from the originally listed 1-level capacity of 1,764 straws to a final capacity of 3,000 straws.
Liquid Nitrogen Cylinders
With the invention of computers, new devices such as computerized controlled rate freezers for cryopreservation of oocytes, semen and embryos (Fig. 13) have been created and developed in order to facilitate these procedures and to secure more control over the techniques. Liquid nitrogen is used to perform the freezing procedures with computerized freezers in order to achieve final temperatures colder than -80°C and ranging between -80°C and -196°C.
Figure 13. IceCube 1810 computerized controlled rate freezer used to cryopreserve semen, embryos and other specimens. On the right of the picture is a 50 liter LN2 cylinder (22 psig) connected to the IceCube freezer through an especially designed connecting hose.
The LN2 can be provided for computerized freezers either in relatively low volume liquid cylinders or through wall outlets that receive the cryogen from centralized storage tanks. Both types of LN2 reservoir can be attached to the freezer by a specially designed connecting hose (Fig. 13).
The LN2 cylinders (Fig. 14) are insulated, pressurized, vacuum-jacketed vessels that are commercially available with volume capacities that can vary from 35 liters up to 450 liters. When compared with ordinary storage tanks, these liquid cylinders are much larger containers. They are mainly used in the workplace where cryopreservation procedures for semen, embryos or other biological specimens are done periodically. LN2 cylinders come equipped with safety relief valves and rupture discs (Fig. 15) to protect them from pressure build-up. These cylinders can operate at pressures ranging between 22 and 350 psi. The pressure required for a computerized freezer such as the ones shown in Fig. 14 is normally 22 psi.
Figure 14. Top: Pictures of two commonly used LN2 cylinders available in the market. Both have a capacity of 50 liters of LN2.
Bottom: Schematic view of the interior parts of a cryogenic cylinder (Picture obtained from www-safety.deas.harvard.edu/services/nitrogen.html).
Figure 15. Top: Schematic top view of different design and configurations of liquid cryogenic cylinders. Middle: Top view of a basic LN2 cylinder with its respective parts.
Bottom: Measuring gauges that are normally found in a LN2cylinder. The left one shows the amount of cryogen left inside the cylinder and the right one the internal pressure. In this case the tank is empty with no pressure inside. (Top picture obtained from www-safety.deas.harvard.edu/services/nitrogen.html).
Centralized storage tanks are enormous mega containers that have a capacity to provide a cryogen to many users located in several different rooms or laboratories at the same time. They can contain between 2,000 and 60,000 liters. These types of tanks can be found in research institutions, hospitals, specialized private companies or universities (Fig. 16).
Figure 16. Centralized cryogen storage tank for oxygen in use at a hospital.