Frozen Semen Packaging Methods

Frozen Semen Packaging Methods in Artificial Insemination (AI)

Frozen semen packaging methods refer to the various techniques used to preserve and store semen at ultra-low temperatures. The early storage of frozen semen was done with glass ampoules in USA.

Japanese scientists later developed the freezing of semen in pellet form. French, Danish and German workers developed the straw technique of frozen semen storage.

Apart from these techniques, the frozen semen was stored in pipettes, bulk forms, sausages, gelatin capsules but they have not become popular because they are not economically feasible and the fertility has been poor.

Different packaging methods of frozen semen include:

  1. Ampoule method
  2. Pellet method
  3. Tupol method
  4. Straw method

1. Ampoule Method

Method of Freezing

  • Ampoule method was developed in USA by Macpherson (1954), Vandemark and Kinney (1954).
  • Immediately after equilibration the semen of o.5-1.0 ml is placed in previously marked glass ampoules.
  • These ampoules were sealed over flame leaving 0.5 ml air space.
  • The ampoules are placed in ethyl alcohol or acetone bath at 5°C.
  • Small quantity of solid carbon dioxide the temperature was brought from 5°C to -15°C (at the rate of 1-2°C/min).
  • Now more solid carbon dioxide is added to bring the temperature to -79°C (at the rate of 4-5°C/min).
  • Six to eight ampoules are arranged in clips or metal cane and each cane is marked with identification of individual bull.
  • Then the ampoules are stored in solid carbon dioxide at -79°C or in liquid nitrogen at -196°C.

Thawing

The ampoule is thawed in iced water for 5-7 minutes. The seal was broken and the semen is aspirated in glass catheter for insemination.

Advantages

  • Hence the semen is stored in the sealed ampoule the chance of contamination is less.
  • Identification is easy as the details are printed over the ampoule and the cane.

Disadvantages

  • As the semen is frozen in larger volume, the freezabilty and fertility is less.
  • The ampoules occupies more space in the cane.
  • About 8-10% of the semen is lost during handling, thawing and inseminating.
  • The insemination is done with glass catheters which is having disadvantages.

2. Pellet Method

Pellet method was developed by Nagase and Niwa (1963) in Japan. Here the semen is rapidly frozen as small pellets on solid carbon dioxide following dilution with media containing carbohydrate and glycerol.

Freezing method

The media used for pellet method is as follow:

ComponentsQuantity
Lactose75.30 ml
Glycerol4.7 ml
Egg yolk20 ml
  • Antibiotics as per the schedule
  • The semen is diluted with the above diluents in a glass tube.
  • It is placed in 250 ml beaker having water at 30°C.
  • The beaker along with the semen is placed in a refrigerator/cold handling cabinet at 5°C for 5 hours.
  • 0.1-0.2 ml of semen is taken in a calibrated pipette and is dropped on solid carbon dioxide.
  • To facilitate this, the solid carbon dioxide was smoothened to a flat surface with an electric sander and small depressions were made by the use of metal di.e.
  • The depressions were approximately 2 mm in diameter and 1 mm in deep.
  • The semen is allowed for 10 minutes for freezing on the solid carbon dioxide.
  • Then they are collected by using pre cooled forceps in to a goblet and stored in liquid nitrogen.

Thawing

The pellet is thawed by placing it in 1 ml 3% sodium citrate solution containing 1.5% fructose at 20°C. The dilute semen is taken in glass catheter and inseminated.

Advantages

  • Economical method
  • Requires less storage space

Disadvantage

  • As the pellets are stored directly identification is difficult
  • As the pellet is placed directly in to liquid nitrogen the chance of contamination with organisms in liquid nitrogen is more
  • When the pellets are taken with forceps for storing and during thawing they may break and gets attached to forceps or may fall anywhere. This leads to loss of sperms.
  • The freezability of sperms by this method is moderate.
  • The thawing is a tedious process when compared to other methods.
  • The insemination is done with glass catheters which is having disadvantages.

3. Tupol Method

  • Roy (1974) used polythene tubes named “Tupol” for freezing semen at -196°C.
  • One end of the tube is immersed in the diluted semen and the other end is attached with the syringe and the semen is sucked into the tube.
  • Then 3-4 inches pieces are cut with electrically heated copper with which seals the cut ends.
  • Insemination is done by cutting one end of the tube and taking the semen in the syringe or glass catheter as per the liquid semen insemination.
  • There is no measure to keep the length of the tube uniform and the sealing is not very satisfactory.
  • More over sealing is done by means of heat, so certain numbers of sperms are damaged at both ends.
  • Therefore volume of semen and number of sperm per dose of insemination is affected.
  • However this method is used in places where other facilities are not available.

4. Straw Method

  • The straw method was introduced by scientist of Denmark during 1940 for packing of liquid semen.
  • Adler (1960) first frozen the semen packed in straws by using liquid nitrogen vapour.
  • This technique was later modified by Cassou in 1965 which was called as “medium straws”.
  • This was in use for a period of time. Later Cassou in 1968 brought further improvement by reducing diameter for better freezing which is called as “mini straws”.
  • Most of the straws recently used were called as “French straws”- made of poly vinyl chloride.
  • Later in German, Simmet (1972) introduced a new straw called “mini tube” or “German straws” or “Landshut system”.
  • Both the ends of this straw was sealed with either steel, glass or plastic beads.
  • For insemination, special sheath is required by which the ball and mini tube can be retained in the sheath allowing only the semen to enter the uterus.
  • Another straw known as “Continental straws” made of polypropylene was developed in United States.

The dimensions of French and German straws are as follows:

Dimensions of French and German Straws
Dimensions of French and German Straws

The French straws have two ends-factory seal end and laboratory seal end. In this the factory seal end was formed in the manufacturers themselves.

The laboratory seal end will be open when it is purchased. The laboratory seal has to be created in the semen processing laboratory after filling the straw with diluted semen before freezing.

For creating laboratory seal earlier the poly vinyl alcohol was used. Recently by using filling and sealing machine, after filling by pressing the straw mechanically the laboratory seal is created.

Advantages of Straw Method

  • Semen is processed as thin film with its greater surface area to volume ratio. Here there is rapid exchange and better revival rate.
  • Reduced volume of the semen is better tolerated by uterus.
  • The diameter of the artificial insemination gun is small so it is easy to pass it through cervix when it is in heat.
  • Complete delivery of semen into uterus is ensured.
  • Identification is easy by using different colour straws, different colour poly vinyl alcohol powder for different breeds.
  • Details like Bull name/No., Breed, Date of collection/batch No. Name of semen bank, first or second ejaculate and other details if required can be printed on the straw.
  • Use of steel gun is safety when compared to glass catheter.
  • The disposable plastic sheath used to cover the AI gun can prevent the disease transmission.
  • Straws occupies less storage space when compared to ampoules.
  • Since the straws is handled with automatic machines the hygiene is ensured.

Method of Freezing

The introduction of freezing of semen led to the better possibility of international exchange of semen. It is possible to use any kind of superior bull semen at any point of time.

The technique of freezing semen helped in the great development of artificial insemination industry.

The advantages of frozen semen are:

  • It permits maximum utilization of semen collected from a sire.
  • Long term preservation of semen for any time period is possible.
  • Semen can be used even after death of the bull.
  • Transport of semen is easy even to international level.
  • Transport cost is frozen semen is less when compared to liquid semen.
  • Selective breeding is possible as per the wish of the owner.
  • It eliminates the maintenance of breeding bull in a herd thereby economic benefit to the owner is ensured.
  • Number of bulls required for breeding purpose can be minimized.
  • Aids in progeny testing of bulls.
  • Round the clock facility is possible even at villages.
  • Door step insemination even at remote places is possible.
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