Ambient Temperature Semen Diluents

Ambient temperature semen diluents are types of semen extenders specifically formulated for use and storage at room temperature (approximately 18–25°C). They are designed to preserve sperm viability and motility for short-term use without the need for refrigeration.

1. Illini Variable Temperature (IVT) Diluent
2. Cornell University Extender (CUE)
3. Milovanov’s Method / Carbonate-Phosphate Method
4. Coconut Milk Extender (CME)
5. Caprogen
6. MG32 Extender

1. Illini Variable Temperature (IVT) Diluent

Van Demark and Sharma has evolved this diluent during 1957 at the University of Illinois, USA. They could preserve semen at room temperature with the help of carbon dioxide gas.

Co₂ temporarily immobilizes spermatozoa and thus reduces their metabolism.

The composition of IVT dilutor:

The above contents are dissolved in boiling double/triple glass distilled water. It is cooled to room temperature and is saturated with carbon dioxide by bubbling the gas through it for ten minutes or until the pH reaches to 6.3.

Egg yolk is mixed at 10 percent level and then antibiotics are added. Then the semen is ampouled and sealed. Before placing the ampoules must be flushed with Co₂.

Semen ampoules are maintained best at room temperature of 20 to 25°C and can be stored up to 6 days with 76 percent fertility on non return basis. Ulaganathan (1970) reported that ascorbic acid supplementation (20 mg/100 ml) to the IVT diluent significantly improved the keeping quality of buffalo sperm stored at 21-31°C.

For longer storage of semen this method does not appear satisfactory.

2. Cornell University Extender (CUE)

Foote et al. (1958) at Cornell University developed the following dilutor for the preservation of semen at room temperature (20-24°C).

To this citric acid 0.12% and 2 mg of catalase per 100 ml of dilutor was added. Antibiotics are also added to control the bacteria. Reaction of citric acid and bicarbonate releases CO₂ which helps in prolonging sperm life.

Small quantity of CO₂ is also produced by spermatozoa from the breakdown of glycine via glyoxylate mechanism, and glyoxylate itself is an effective inhibitor of respiration.

Thus, in medium to which glycine and citric acid are added in addition to bicarbonate, there is no need to infuse extra CO₂.

The gas generated in the medium is sufficient to curtail respiration of spermatozoa and immobilize them temporarily. Addition of catalase at room temperature preservation was necessary to act on hydrogen peroxide produced by spermatozoa from amino acids.

Semen can be preserved for 3-6 days with 70-80% fertility on non return basis.

3. Milovanov’s Method

Milovanov’s method is also called as “carbonate phosphate method”. It was developed by Milovanov (USSR) during 1959.

Composition of this media is as follows:

Part A and Part B

Mix Part A and Part B in the ratio of 1:9.

Part C

• Add 11 ml of egg yolk to the above solution (AB).
• The antibiotics are added as per the schedule.
• One ml of the diluted semen are sealed off in glass ampoules and stored at 20-25^oC.

4. Coconut Milk Extender (CME)

Charl’s Norman et al. (1958) identified that the coconut milk can be used for semen preservation. This media was later improved by adding catalase, polymyxine and mycostatin.

The composition is as follows:

Part A

Part B

• Coconut milk (water)-15 ml
• Boil the coconut milk for 10 minutes, filter and add to Part A.

Part C

• Egg yolk: 7 ml
• Add this egg yolk to AB. Bring the volume to 100 ml using distilled water. Adjust the pH to 7.4
  • The vials are filled without air space.
  • Wrap it with aluminium foil.
  • Store at dark place
  • During transport use insulated bag to avoid adverse effects
• Norman (1968) used this dilutor for buffalo semen and could preserve buffalo semen for seven days at room temperature.
• Coconut milk has the following chemical composition

This extender is not commercially successful due to the non-availability of the good quality coconut throughout the year.

5. Caprogen

Caprogen was developed in New Zealand. The initial development of this extender for 5:C storage was modified by adding 2-5 percent egg yolk and catalase to decompose the H₂O₂ that is formed as a product of sperm metabolism.

The optimum temperature range for caprogen is considered to be 18-24:C.

6. MG32 Extender

The composition of the diluent is given below:

MG32 extender was developed by Ramamurthy during 1973. It used successfully in buffalo semen.