Thawing, Loading, and Sites of Semen Deposition

Thawing, loading, and semen deposition sites are key steps in artificial insemination. Thawing warms frozen semen to revive sperm, usually at 37°C for about 30 seconds. Loading involves placing the thawed semen into an AI gun under sterile conditions. The site of semen deposition varies by species—for example, the uterus in cows and the cervix or uterus in sows—to ensure the best chance of fertilization.

The frozen semen are stored in different types of straws. They are:

• French medium straw (0.5 ml)
• French mini straw (0.25 ml)
• German mini tube (0.25 ml)

Thawing French Semen Straw

• Lift the neck plug of the container
• Pre cool the forceps in the liquid nitrogen vapour for 5-10 seconds
• Lift the canister from the neck and move it to its opposite edge. Now the body of the canister will come to centre.
• Lift the canister to the level of neck
• Identify and lift the straw by forceps
• The whole operation of lifting of canister and removal of straw should be done as fast as possible i.e. within 5-10 seconds. Otherwise immerse the canister into the liquid nitrogen.
• Immediately after removing the semen straw give a jerk to remove the liquid nitrogen on the straw and also to avoid popping of the seal.
• If the straw is having cracks or broken or defective seals the straw should be discarded.
• Put the semen straw in a water bath of 37:C for 30 seconds. While thawing the semen straw should be completely submerged in water.
• The thawing water should be fresh and clean. The container used to hold the thawing water should also be clean.
• Thaw the semen straw either in horizontal or vertical manner
• After the thawing, remove the straw and wipe with cotton to remove all the water on the surface of the straw.

Loading of AI gun (French Straw)

• Use appropriate insemination gun for respective semen straws or use universal gun to load the straws.
• By gently tapping the straws bring the air space near to laboratory seal.
• Place the straw in the chamber of the insemination gun. While placing the factory seal should go down and the laboratory seal should come up.
• Cut the air space area of the straw by keeping the straw in upright position.
• While cutting it should not be cut too close to the semen or should not be cut at angles which will lead to back flow of the semen or wastage of the semen at the time of insemination.
• Fit the sterile sheath over the straw and the chamber of the inseminating gun.
• Obtain perfect fit between extremity of the straw and the cone of the sheath.
• The sheath should be fixed with the button of the inseminating gun.
• In Universal AI gun auto lock is available which will hold the sheath firmly.
• After placing the sheath, do not touch the tip of the sheath. Handle only the base of the sheath.
• The post thaw survival of spermatozoa is poor.
• For maximum reproductive efficiency thawed semen should be used immediately.
• So do not thaw more than one straw at one time.

Site of Semen Deposition

• Frozen thawed semen is deposited in internal os of cervix or body of uterus.
• Avoid depositing in horn of the uterus.

Inseminating with German Minitube Straw

• For German minitup straws the gun and sheath are different than French gun and sheath.
• The gun is made of stainless steel rod of 18 inch long and the sheath has special arrangement at its tip and is called as plug tipped sheath.
• The minitup after cutting at one end is inserted in the sheath (cut end downward) and then gun is fitted.
• At the time of insemination, the semen is deposited by pressing the gun and the sealing ball is retained at the tip of the sheath.
• After insemination it is taken out and the sheath along with the empty minitup are discarded.
• By this method with German gun medium French straw can also be inseminated but with the French gun minitup cannot be inseminated.

Thawing Semen From Ampoules

• The ampoule is taken out from the liquid nitrogen container carefully.
• Thaw the ampoules in iced water for 6-7 minutes.
• Wipe the ampoule with absorbent cotton.
• Cut and draw the semen in a sterilized catheter fitted to a plastic/glass syringe with rubber connector.
• Inseminate the animal using speculum.

Precautions to be taken While Inseminating a Cow

• The AI gun should be sterile.
• Thaw the semen under hygienic conditions using fresh, warm water.
• Introduce the AI gun at an angle of 45: initially, and then horizontally glide along the dorsal side of the vagina.
• Never force the gun when it is within vaginal folds which will leads to injury to the tract.
• Don’t inseminate the cow when it is not in heat.
• Remember that poor fertility with frozen semen is due to improper handling or deposition of semen or both.