Sterilization of Artificial Insemination (AI) Laboratory Equipment

Sterilization of Artificial Insemination (AI) laboratory equipment is the process of completely removing all microorganisms, including bacteria, viruses, fungi, and spores, from the tools and materials used in semen collection, processing, and insemination.

This process ensures the cleanliness of the laboratory environment, prevents contamination of semen samples, maintains sperm quality, and follows standard protocols for reproductive procedures in animals.

An appropriate method of sterilization depends on the properties of the material. Immediately after use, all glass wares, rubber wares, artificial vagina, etc. should be immersed in mild neutral soap solution to check the growth of microbes.

After the collection of semen, these materials should be washed in running tap water and the sterilization should be done according to the material and its use.

Artificial Vagina (AV)

• Cone from the AV and water from the Artificial Vagina (AV) jacket should be removed before washing.
• Cones and AVs are cleaned thoroughly with soft sponge brush under running tap water and then soaked in warm neutral cleanser for about 30 minutes, followed by proper rinsing in warm and clean water and lastly washed three times with double distilled water.
• For sterilization, fully assembled AVs should be autoclaved at 5 psi pressure for 20 minutes.
• During sterilization the AV valve should be kept open.
• Finally, the AVs should be stored in the incubator at around 40-50°C for next few days use.
• To achieve best cleaning effect, AVs should be cleaned immediately after use, preferably by non-spermicidal neutral detergent.

Glass Wares

• The glass wares (conical flask, measuring cylinder, beaker etc.) have to be immersed in neutral detergent solution immediately after their use.
• They should be washed thoroughly in running tap water and soaked in warm, non- spermicidal detergent solution for about 30 minutes.
• Subsequently these are thoroughly brushed with appropriate nylon brush, washed in running tap water and finally again three times with double distilled water.
• The washed glass wares were allowed to dry by keeping them on inverted position on a blotting paper.
• Dried glass wares were covered with aluminium foil and sterilized in hot air oven at 160°C for 1 hour.

Collection Tube

• The semen collection tube/vial should be immersed in a neutral detergent solution immediately after its use.
• It should be washed thoroughly with running tap water and soaked in warm, non- spermicidal neutral detergent solution for about 30 minutes.
• Using appropriate nylon brush, the collection tube should be cleaned in all the sides and bottom to remove the semen particles and rinsed with running tap water.
• Finally, the collection tube should be rinsed three times with double distilled water and allowed to dry in oven at 60°C.
• The open end of the dried collection tube should be closed with aluminium foil an sterilized in hot air oven at 160°C for 1 hour or at 180°C for 30 minutes.

Rubber Wars

• The washing and cleaning procedure of rubber wares is similar to that of glass wares.
• Care should be taken to clean the rubber wares with sponge brush instead of nylon brush.
• Plastic tips and tubing’s should be cleaned by water jet with force using a suitable syringe.
• Sterilization technique, however, differ owing to the thermo sensitivity of the rubber items.
• Thermo sensitive rubber wares are packed and sealed in specific polythene bags and sterilized in ethylene oxide gas sterilizer.
• Thermo resistant rubber wares are to be sterilized by autoclaving at 3-4 psi for 10 minutes.
• The sterilized rubber wares should be kept in an UV chamber.
• The rubber tube used for semen filling should not be reused.

Distilled Water

• Triple distilled or Milli-Q purified water should be used for preparation of dilutor.
• The distilled water should be sterilized by autoclaving at 15 psi for 15 minutes.
• For storing the distilled water autoclaved sterile glass wares should be used.
• The triple distilled or Milli-Q water stored for more than one week should not be used.

Buffer

• The buffer can be prepared in the previous day evening of the collection.
• The chemicals used for buffer preparation should be Graded reagent (GR) or Analytical grade (AR) grade and should be purchased from standard companies.
• Laboratory grade (LR) chemicals should not be used due to low purity.
• Whenever new chemicals are purchased it is recommended to conduct the post thaw survival rate by conducting few split ejaculate trials (maintaining the previous batch as control) with the new chemical.
• Buffer should be prepared in a sterile manner in the Buffer and Diluent preparation room.
• Buffer should be sterilized by autoclaving at 5 psi pressure of 20 minutes.
• Upon cooling and after addition of egg yolk, antibiotics should be added under laminar flow unit.
• The buffer should not be stored and reused in subsequent days.

Diluent

• The dilutor should be prepared fresh on the day of semen collection early in the morning.
• Sometimes it can be prepared in the previous day evening but it should be noted that the antibiotics should be added only in the morning prior to use.
• Diluent should be prepared under Laminar Airflow Unit (LAFU).
• Egg purchase, storage and the egg yolk preparation should be done as per standard procedure.
• After adding the egg yolk to the buffer, it should be stirred for 30 minutes for complete mixing of egg yolk to the buffer.

Bacteriological Media

• Standard plate count agar is used for bacteriological examination of the fresh, frozen semen and also for the consumables used in semen bank.
• The media is autoclaved at 15 psi pressure for 15 minutes.
• The entire procedure should be done under Laminar Air Flow Unit to avoid false positive results.

Filter Papers

• Filter papers of approximately 15×10 cm size are to be used for egg yolk separation.
• A bunch of such papers are thrashed to remove dirt, if any and wrapped in thick cotton cloth for sterilization in autoclave at 5 psi pressure for 20 minutes.
• The autoclaved filter papers can be kept under laminar air flow and can be used for diluent preparation.
• It is better to purchase Whatman No.1 filter paper for this purpose.

Summary of Sterilization

Autoclave

Hot Air Oven

Item	Temperature	Time (Min.)
Glass wares	160°C / 180°C	60/30
Filling nozzles	160°C / 180°C	60/30

Ethylene Oxide Gas Sterilizer

• The exposure time for gas sterilization is inversely proportional to the gas concentration under which the equipment is exposed in the sterilizer.
• Commonly used concentration of ethylene oxide is 900 mg/litre of cubic space for a period of 6 hour.
• All items sterilized with gas must be ventilated adequately before use.
• A minimum of 72 hours at room temperature with adequate ventilation is recommended.
• Items to be sterilized are tubings of filling and sealing machine, rubber plugs of filling nozzles.