Technical cornerstones of successful AI in cattle
There is a plethora of variables that play into the success or failure of artificial insemination (AI). From health and nutrition status of the sires, to frequency of semen collection, all the way to the right point in time in the cow’s estrus, countless parameters must be considered.
One critical stage, however, is the storage and handling of semen doses prior to insemination. Semen is highly sensitive even to seemingly harmless environmental influences, such as temperature fluctuations or even plain water. Most inseminations in cattle are conducted using frozen-thawed semen filled in 0.25 ml or 0.5 ml straws. While freezing semen extends storage time by decades, freezing and thawing semen are highly delicate processes that require knowledge, experience, and specialized high-quality equipment.
This article aims to provide an overview of all the factors that need to be considered to maintain semen quality from storage in liquid nitrogen tanks, to thawing of semen doses all the way to the preparation of the insemination device.
Consisting of two layers, the inner chamber of the tank containing the liquid nitrogen is insulated by the vacuum filled outer chamber. The outside shell of liquid nitrogen tanks should not feel overly cold to the touch, nor should it have an accumulation of frost at the neck. These are telltale signs of rapid evaporation of liquid nitrogen, most likely caused by a crack in the shell and resulting vacuum loss. In this case, semen doses should be transferred to a backup nitrogen container as soon as possible.
To avoid damages to the container in the first place, the tank needs to be handled with care and must not be subjected to sudden, jarring movements or collisions with other objects. In addition, liquid nitrogen tanks need to be protected from humidity to avoid corrosion of the shell. The containers should be stored in a clean, dry, dust-free area with good light conditions, but without direct sunlight. Regular maintenance encompasses monitoring liquid nitrogen levels (levels should not drop below 8 cm/3 inches) and keeping a record of nitrogen loss. The easiest way to do this is using a measuring stick.
For shipment of frozen semen, containers that comply with international regulations must be used. In many cases, such containers must not contain liquid nitrogen. Vapor Shippers such as the MVE Vapor Shippers or the CryoCube™ Dry Shipper are good options. The nitrogen vapor in these containers ensures transport of semen doses at temperatures below -150°C (-238°F) for a minimum of five days.
But to be able to grasp and identify single semen doses, the canister needs to be raised up to the neck of the tank, where temperatures surpass the aforementioned -80°C (-112°F) that represent the threshold towards temperatures that are critically dangerous to sperm cells. Picture 1 shows the exact temperature distribution [°F] in the neck of a liquid nitrogen tank (adapted from Saacke, 1974).
The most important measure for protecting semen doses from prolonged exposure to dangerously high temperatures is having a well-maintained inventory system in place that lists the content of every canister, ideally combined with color-coded goblets. That way, the time spent searching for a particular semen dose can be minimized.
When retrieving a semen dose, the canister should be kept as low in the tank as possible and only raised above the frost line for a maximum of 10 seconds. If necessary, the canister should be lowered back into the tank for 10-15 seconds to cool back down before trying again. In the same vein, semen straws in the liquid nitrogen tank should always be handled with tweezers, not with bare hands to avoid additional heat exposure. Generally, in the handling of semen doses, a quick and organized mode of operation is key.
Preferably, a thawing device such as the MiniThaw should be used for thawing semen doses. The small, lightweight device can be filled with water and heated in advance, is lockable and guarantees the exact right water temperature.
There is, however, a caveat to warm water thaw: Since sperm cells can be permanently damaged by substantial temperature fluctuations, temperature management needs to be monitored closely. The straws must not be left in the warm water for longer than the recommended 25-30 seconds for 0.25 ml straws or 30 seconds for 0.5 ml straws so that semen temperature does not raise above approximately +22°C.
In the same vein, precautions need to be taken to avoid a sudden drop in temperature of semen doses post-thaw. This includes working at ambient temperature, prewarming the insemination device and keeping it warm during the transport to the cow. If these points are taken into consideration, warm water thaw is significantly superior to other thawing methods. Given that water temperature remains constant, and water is agitated repeatedly to avoid freezing together, up to 10 to 20 straws can be thawed at once. It is, however, not advisable to thaw more semen doses in one round than can be inseminated within 10 to 15 minutes, otherwise semen quality will suffer.
In order to obtain optimal insemination results, it is advisable to purchase all components of AI equipment from one reliable supplier of high-quality products. That way, the single items will be coordinated exactly and facilitate a smooth operation.
Also, only an AI gun that is suitable for the cow as well as ergonomic for the operator will provide optimal results. In that vein, Minitube’s QuickLock series offers three different types of AI guns (QuickLock Classic, QuickLock Ergo, QuickLock 2000) that cover a range of preferences. The Universal sheaths, available as split or non-split, are coordinated optimally with all three types of QuickLock AI guns to ensure a perfect fit. Picture 2 shows the perfectly tight fit between sheath, AI gun and straw opening.
Tip: Sheaths must not be subjected to direct sunlight or heat for extensive periods of time. This can result in shortening or deformations that might not even be visible but can impair the sealing of the sheath with the straw and/or the AI gun.
To prepare the straw for the insertion into the AI gun after it is taken out of the water bath, first it needs to be dried off completely since water it lethal to sperm. Then, after making sure the air bubble is at the heat-sealed end, the straw is cut below the sealing and subsequently loaded into the insemination device with the cotton-plug-end first. Using a specialized device such as the MiniCutter ensures a clean, straight cut, which in turn diminishes the risk of retrograde flow of semen in the AI instrument during insemination.
Finally, a plastic AI sheath is placed over the insemination gun. Investing in high-quality sheaths is worthwhile since cheap sheaths can also be a reason for retrograde flow of semen in the AI gun. Straws often don’t seal properly with the exit opening of low-quality sheaths and, therefore, the semen will flow inside the sheath when deposited and get lost. After mounting the sheath, the plunger of the readied insemination instrument is pushed until all air has left the straw.
Following the assembly of the AI device, the insemination should be conducted as soon as possible. For the transport to the cow, the instrument should ideally be stored in the QuickLock Heater 4.0.
Tip: AI equipment should be cleaned with 70% isopropyl alcohol to avoid cross-contamination. Disinfectants and soaps are highly damaging to sperm cells and are therefore not suited as a cleaning agent.
An important factor in providing as much comfort for the animal as possible is using generous amounts of sperm friendly lubricant for the rectalization, such as ReproJelly.
Literature cited in this article:
Concepts in semen packaging and use; Saacke, R. G. 1974
Effect of Cold Shock after Thawing on Acrosomal Maintenance and Motility of Bovine Spermatozoa Frozen in Plastic Straws; Almquist, J.O. 1976
Influence of thawing temperature on sperm motility, structure, and metabolism of frozen bovine semen; Zenteno, E.S., Rojano, B., Betancur, G. R. 2023
Proper Semen Handling Improves Conception Rates of Dairy Cows; Looper, M. 2000
Semen handling Checklist – GENEX
Storing and handling frozen semen; O’Connor, M. L. 2016
One critical stage, however, is the storage and handling of semen doses prior to insemination. Semen is highly sensitive even to seemingly harmless environmental influences, such as temperature fluctuations or even plain water. Most inseminations in cattle are conducted using frozen-thawed semen filled in 0.25 ml or 0.5 ml straws. While freezing semen extends storage time by decades, freezing and thawing semen are highly delicate processes that require knowledge, experience, and specialized high-quality equipment.
This article aims to provide an overview of all the factors that need to be considered to maintain semen quality from storage in liquid nitrogen tanks, to thawing of semen doses all the way to the preparation of the insemination device.
Semen storage and transport in nitrogen containers
To preserve semen quality parameters as much as possible in frozen semen, semen doses should be stored at temperatures well below -80°C (-112°F). Up until this temperature, the ice crystals formed from the water content of the semen and extender are not completely stable and pose a threat to the sperm cells. For this reason, it is common practice to store sperm doses in liquid nitrogen containers, within which the temperature is a constant -196°C (-321°F).Consisting of two layers, the inner chamber of the tank containing the liquid nitrogen is insulated by the vacuum filled outer chamber. The outside shell of liquid nitrogen tanks should not feel overly cold to the touch, nor should it have an accumulation of frost at the neck. These are telltale signs of rapid evaporation of liquid nitrogen, most likely caused by a crack in the shell and resulting vacuum loss. In this case, semen doses should be transferred to a backup nitrogen container as soon as possible.
To avoid damages to the container in the first place, the tank needs to be handled with care and must not be subjected to sudden, jarring movements or collisions with other objects. In addition, liquid nitrogen tanks need to be protected from humidity to avoid corrosion of the shell. The containers should be stored in a clean, dry, dust-free area with good light conditions, but without direct sunlight. Regular maintenance encompasses monitoring liquid nitrogen levels (levels should not drop below 8 cm/3 inches) and keeping a record of nitrogen loss. The easiest way to do this is using a measuring stick.
For shipment of frozen semen, containers that comply with international regulations must be used. In many cases, such containers must not contain liquid nitrogen. Vapor Shippers such as the MVE Vapor Shippers or the CryoCube™ Dry Shipper are good options. The nitrogen vapor in these containers ensures transport of semen doses at temperatures below -150°C (-238°F) for a minimum of five days.
Retrieval of semen doses from the liquid nitrogen container
Retrieving semen doses from the liquid nitrogen tank is a critical step in the preparation of an insemination that can jeopardize not only the quality of the semen dose retrieved, but also the quality of every other semen dose in the canister. Given their large surface-to-volume ratio, semen in straws can be negatively affected by too high temperatures within seconds of exposure.But to be able to grasp and identify single semen doses, the canister needs to be raised up to the neck of the tank, where temperatures surpass the aforementioned -80°C (-112°F) that represent the threshold towards temperatures that are critically dangerous to sperm cells. Picture 1 shows the exact temperature distribution [°F] in the neck of a liquid nitrogen tank (adapted from Saacke, 1974).
The most important measure for protecting semen doses from prolonged exposure to dangerously high temperatures is having a well-maintained inventory system in place that lists the content of every canister, ideally combined with color-coded goblets. That way, the time spent searching for a particular semen dose can be minimized.
When retrieving a semen dose, the canister should be kept as low in the tank as possible and only raised above the frost line for a maximum of 10 seconds. If necessary, the canister should be lowered back into the tank for 10-15 seconds to cool back down before trying again. In the same vein, semen straws in the liquid nitrogen tank should always be handled with tweezers, not with bare hands to avoid additional heat exposure. Generally, in the handling of semen doses, a quick and organized mode of operation is key.
Thawing semen
When it comes to thawing semen doses, it is crucial to minimize the time of exposure to harmful temperature ranges and thus allow the semen to thaw very quickly. Therefore, optimal sperm cell recovery is achieved when thawing semen doses in warm water of about 32-40°C (90-104°F) for 25-30 seconds (0.25 ml straws) or 30 seconds (0.5 ml straws). Practices such as thawing semen doses in your pocket or even in the cow are not advisable since thaw rates are too slow and therefore damaging to the semen.Preferably, a thawing device such as the MiniThaw should be used for thawing semen doses. The small, lightweight device can be filled with water and heated in advance, is lockable and guarantees the exact right water temperature.
There is, however, a caveat to warm water thaw: Since sperm cells can be permanently damaged by substantial temperature fluctuations, temperature management needs to be monitored closely. The straws must not be left in the warm water for longer than the recommended 25-30 seconds for 0.25 ml straws or 30 seconds for 0.5 ml straws so that semen temperature does not raise above approximately +22°C.
In the same vein, precautions need to be taken to avoid a sudden drop in temperature of semen doses post-thaw. This includes working at ambient temperature, prewarming the insemination device and keeping it warm during the transport to the cow. If these points are taken into consideration, warm water thaw is significantly superior to other thawing methods. Given that water temperature remains constant, and water is agitated repeatedly to avoid freezing together, up to 10 to 20 straws can be thawed at once. It is, however, not advisable to thaw more semen doses in one round than can be inseminated within 10 to 15 minutes, otherwise semen quality will suffer.
AI equipment
In order to obtain optimal insemination results, it is advisable to purchase all components of AI equipment from one reliable supplier of high-quality products. That way, the single items will be coordinated exactly and facilitate a smooth operation.Also, only an AI gun that is suitable for the cow as well as ergonomic for the operator will provide optimal results. In that vein, Minitube’s QuickLock series offers three different types of AI guns (QuickLock Classic, QuickLock Ergo, QuickLock 2000) that cover a range of preferences. The Universal sheaths, available as split or non-split, are coordinated optimally with all three types of QuickLock AI guns to ensure a perfect fit. Picture 2 shows the perfectly tight fit between sheath, AI gun and straw opening.
Tip: Sheaths must not be subjected to direct sunlight or heat for extensive periods of time. This can result in shortening or deformations that might not even be visible but can impair the sealing of the sheath with the straw and/or the AI gun.
Loading the AI gun
To guard against temperature fluctuations, the insemination device should be prewarmed before inserting the semen dose. A convenient way to do this is using a specialized heating bag that can be set to the ideal temperature, such as the QuickLock Heater 4.0.To prepare the straw for the insertion into the AI gun after it is taken out of the water bath, first it needs to be dried off completely since water it lethal to sperm. Then, after making sure the air bubble is at the heat-sealed end, the straw is cut below the sealing and subsequently loaded into the insemination device with the cotton-plug-end first. Using a specialized device such as the MiniCutter ensures a clean, straight cut, which in turn diminishes the risk of retrograde flow of semen in the AI instrument during insemination.
Finally, a plastic AI sheath is placed over the insemination gun. Investing in high-quality sheaths is worthwhile since cheap sheaths can also be a reason for retrograde flow of semen in the AI gun. Straws often don’t seal properly with the exit opening of low-quality sheaths and, therefore, the semen will flow inside the sheath when deposited and get lost. After mounting the sheath, the plunger of the readied insemination instrument is pushed until all air has left the straw.
Following the assembly of the AI device, the insemination should be conducted as soon as possible. For the transport to the cow, the instrument should ideally be stored in the QuickLock Heater 4.0.
Tip: AI equipment should be cleaned with 70% isopropyl alcohol to avoid cross-contamination. Disinfectants and soaps are highly damaging to sperm cells and are therefore not suited as a cleaning agent.
Animal welfare
To keep stress and discomfort of the cow to a minimum, the animal needs to be handled gently and calmly. In addition to causing unnecessary stress to the animal, undue agitation or rough handling can also lead to diminished semen transport in the animal’s reproductive tract and thus to lower conception rates.An important factor in providing as much comfort for the animal as possible is using generous amounts of sperm friendly lubricant for the rectalization, such as ReproJelly.
Literature cited in this article:
Concepts in semen packaging and use; Saacke, R. G. 1974
Effect of Cold Shock after Thawing on Acrosomal Maintenance and Motility of Bovine Spermatozoa Frozen in Plastic Straws; Almquist, J.O. 1976
Influence of thawing temperature on sperm motility, structure, and metabolism of frozen bovine semen; Zenteno, E.S., Rojano, B., Betancur, G. R. 2023
Proper Semen Handling Improves Conception Rates of Dairy Cows; Looper, M. 2000
Semen handling Checklist – GENEX
Storing and handling frozen semen; O’Connor, M. L. 2016
