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STORING AND HANDLING FROZEN SEMEN

Michael O’Conner
Pennsylvania State Univ.

Considerable change has occurred within the artificial insemination (Al) industry over the past ten years. The most obvious change has been the transition from the ampule to the straw as the predominant semen package. This package system has several advantages over the ampule. More units can be stored in bulk at the AI organization and in farm semen tanks. The straw system also allows more complete delivery of semen during insemination. Probably most important, the straw permits more uniform control of the freezing and thawing process which has led to improved sperm cell recovery. The major disadvantage of the straw system is vulnerability to mishandling.

In addition to the adoption of the straw there has been an increase in direct sales of semen to owner-inseminators. It's estimated that the average farm semen tank contains semen from two to three breeding organizations. Although the .5 ml French straw is the most popular semen package available today, .5 ml and 1 ml ampules are still present in some tanks. The .25 ml straws may be available in some areas. Each Package system has a different surface to­-volume ratio which requires unique handling procedures. Recommendations for handling semen also vary among AI organizations using the same package. This whole set of circumstances has resulted in confusion for owner-inseminators and, in some cases, fostered the notion that almost any method of handling semen is adequate. Whether this attitude comes from indifference, ignorance, or confusion, the end result may be lowered conception rate.

Semen tank management

Technical advances have been made on the design and construction of semen tanks. Tanks with six- to eight-months liquid nitrogen holding times are available. The maintenance of very low liquid nitrogen temperatures in the inner chamber is due to high quality solid insulation material and vacuum in the outer chamber. Although the newer tanks are better insulated, they are still susceptible to damage from mishandling. The inner chamber containing liquid nitrogen is actually suspended from the outer shell by the neck tube. Abnormal stress on the neck tube caused by sudden jarring or excessive swinging motion could crack the tube and result in vacuum loss. Since vacuum is the major insulation component of the tank, a loss of vacuum causes an increase in temperature within the inner chamber and a rapid evaporation of nitrogen. Accumulation of frost at the top of the tank indicates a rapid evaporation of liquid nitrogen.

Several field studies have indicated that there is no significant damage to semen stored in properly managed farm tanks.

However, it should be noted that during a field study in the state of Washington, liquid nitrogen completely evaporated in three of 60 tanks. This may appear to be a low percentage, but it is a significant economic loss to the individual producer.

To avoid such a tragedy, follow these simple management practices:

1. Avoid excessive movement or abuse of the tank.

2. Monitor nitrogen levels routinely and keep a record of nitrogen loss. Remember, even new tanks can have defects and fail.

3. Store the semen in an area with good light but out of direct sunlight. Observe the tank daily. Once a tank fails, nitrogen is lost very rapidly. Have an alternative semen tank available in case of a failure.

4. Keep the tank elevated above the concrete floor or other wet and poorly ventilated surfaces. Corrosion of the outer shell will shorten the functional life of the tank and possibly cause failure.

5. Store only the amount of semen needed for six months.

Handling semen within the tank

When extended semen cools during the freezing process, microenvironments are created within the semen package. Each chemical component of extended semen freezes or solidifies at a different temperature. Water freezes as temperatures drop below 32ºF forming ice crystals which remain somewhat unstable at temperatures above 112º F. This instability may be due to recrystallization of the ice. Also, as water is converted to ice, the sperm are exposed to the remaining concentrated solution of salts and other components of extender which freeze at temperatures considerably below the freezing point of water. Instability of ice and concentrated solutions are harmful to sperm. Fortunately, incorporating glycerol as a cryoprotective agent and improving freezing rates have minimized sperm damage. However, semen must be kept at temperatures well below critical temperatures where the recrystallization of ice begins to occur.

In the typical farm semen tank, dangerous temperatures exist in the upper half of the neck tube.

Exposure to these temperatures can occur when semen is transferred from tank to tank or when handling semen within the neck while trying to locate and thaw a specific unit of semen. Remember, the larger surface-to-volume ratio of the straw makes it very susceptible to thermal fluctuation.

Thermal injury to sperm is permanent and cannot be corrected by returning semen to liquid nitrogen. The following semen handling practices are recommended to minimize thermal damage:

1. Transfer of semen between tanks must be coordinated and rapid. Two people should be involved, and tanks should be arranged side by side. If possible, fill the tanks with nitrogen before transfer. Raise canisters only to a level necessary to locate the appropriate rack of semen.

2. Develop a semen inventory system and mount it on the wall above the tank. It is best to keep semen from one bull on each rack. Such systems help avoid unnecessary searching and exposure of semen to dangerously high temperatures within the neck region.

3. Prepare to thaw semen by raising the canister into the lower portion of the neck where the desired rack of semen can be grasped. Lower the canister further into the neck. Secure the rack as low as possible in the neck to protect the other straws from thermal damage. If the straw cannot be easily removed from the plastic goblet, bend the top tab of the rack to a 45º angle to reduce the chance of bending the straw.

4. Use tweezers to transfer the straw to the thaw bath. Quickly lower the rack of semen and canister into the tank body.

Thawing semen

When the .5 ml French straw was first introduced in this country, there was confusion about the optimal thawing method. Recommendations varied among AI organizations, each of which has a specific method for diluting, cooling, packaging, and freezing semen in straws. The total processing system determines the optimal rate of thaw. As a result of considerable research, it is generally concluded that warm water thaw (95ºF) results in improved sperm cell recovery compared with other methods of thawing. Success of warm water thaw is due to the fact that sperm are exposed to critically dangerous temperatures for only a brief amount of time. The rise in temperature is rapid enough to minimize sperm damage.

A major criticism and concern for the warm water thaw is the danger of cold shock caused by mishandling the straw following thawing. Cold shock is the permanent injury to sperm caused by a sudden decrease in semen temperature after thawing. It can occur during preparation of the inseminating device or transport to the cow. If precautions are taken to prevent cold shock, the advantage of warm water thaw will be realized.

It is important that the temperature of the thaw water be checked immediately before removing the straw from the tank. Use an accurate, easy-to-read thermometer. The length of thaw should be at least 40 seconds.

Some organizations recommend the pocket thaw for straws. This method is successful for semen processed and packaged by their system. However, the pocket thaw should not be used for semen packaged in straws from other organizations.

The standard 1 ml ampules should be thawed in ice water (41º F) for 10 minutes. The smaller .5 ml ampules can be thawed in warm water for 90 seconds or ice water for three to five minutes. Semen should be thawed according to the recommendations of the organization supplying that specific unit of semen.

Inseminating a group of synchronized cattle

Breeding a group of cattle during a short period of time following estrous synchronization may present problems for the herd manager and Al technician. Although artificial insemination of synchronized cows is the final step in a synchronization breeding program, it is critical to the success of the entire program. It is important that special consideration be given to proper semen handling and insemination technique to ensure optimum conception rate.

1. Personnel: Assign each person a job to move the breeding phase of this program along efficiently. One individual should be responsible for thawing semen and preparing the inseminating gun. This relieves the AI technician of additional tasks, allowing concentration on proper AI technique. Additional people could move cattle to and from the breeding chute. Everyone should be thoroughly instructed in their specific jobs prior to the breeding.

2 . Physical facilities: Ensure an adequate holding area is available where heifers can be assembled for treatment, heat detection, and breeding. This area should have a breeding chute or similar arrangement where animals can be treated and artificially bred in a safe and efficient manner, If possible, the synchronized animals should be observed for heat following hormone treatment. This would require that the holding area be equipped with sufficient feed and watering facilities for approximately five days until all synchronized cows are bred. The breeding chute area should be covered to protect semen, supplies, records, and personnel from adverse weather.

3. Procedures:

A. Prepare a list of selected matings if numerous AI sires are to be used for breeding the synchronized cattle. This list could be used in selecting which unit of semen is to be thawed and inseminated for each particular cow. An inventory system describing the location of semen from each bull within the semen tank is also desirable.

B. Follow thaw procedures according to the recommendations of the AI organization supplying the semen. The thaw water must be maintained at the proper temperature for each dose of semen thawed. NOTE: recent research from Washington State has shown that 10 to 20 straws can be thawed simultaneously as long as the thaw bath water remains a constant 95ºF and the environmental temperature is not severely cold. Straws thawed in bulk should be agitated slightly to keep them from sticking together. Bulk thawing of semen should only be considered when a large group of synchronized cattle are to be inseminated.

C. Do not prepare the insemination gun too far in advance of insemination. Breed the cow as soon as possible after the semen is properly thawed and the inserninating equipment is assembled.

D. Prepare insemination devices in a warm, clean environment near the breeding chute, but far enough away to avoid excessive dust and debris near the cattle. This will minimize the chance of contaminating the equipment and semen.

4. Other considerations:

A. Handle animals gently to avoid unnecessary excitement before, during, and after breeding. Undue excitement may adversely affect sperm transport within the female reproductive system causing a lower conception rate.

B. Breed animals based on standing heat, remembering to breed the animals 10 to 12 hours after the beginning of standing behavior.

C. Use proper insemination techniques. Sufficiently qualified inseminators should be on hand if many cows are to be bred over a short period of time. Consult your Al representative for advice and help in this regard.

The success of a synchronization-breeding program depends on prior planning, teamwork, and attention to detail by everyone involved.

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