Tris–Egg Yolk–Glycerol Diluent (Universal Diluent): Composition and Preparation

Tris–Egg Yolk–Glycerol Diluent, also known as the Universal Diluent. It is a widely used semen extender specifically formulated for cryopreservation of semen at ultralow temperatures (−196°C in liquid nitrogen).

Composition

Inclusion of glycerol at room temperature was found as satisfactory as 5°C. Tris diluter allows filling and sealing of poly vinyl straws at room temperature while freezing the semen.

Preparation of Egg Yolk

• Unfertilized egg is used for this purpose.
• The egg should be purchase from disease free, known flock.
• Fresh egg immediately after laying is purchased
• The egg immediately after receiving is wiped with dry cotton and stored at refrigerator at 4-5°C.
• Maximum it can be stored for 3 days at refrigerator temperature of 4-5°C.
• Immediately before use it is cleaned with swab of 70% alcohol.
• It is opened at the narrow end with the help of a sterilized forceps.
• The white of an egg (albumen) is drained off with the help of an egg yolk separator and yolk is placed on a sterile filter paper.
• If required yolk may be transferred to another filter paper to clear traces of albumin.
• Then the vitelline membrane is punctured with a sterile glass rod and yolk is allowed to fall in a sterilized graduated cylinder directly at the bottom without touching the walls.
• The required quantity is prepared and added with autoclaved buffer (added with glycerol) and mixed with magnetic stirrer.

Diluents Preparation

The diluents preparation involves two steps:

1. Preparation of buffer
2. Preparation of diluents

Points to be considered before preparation of diluent:

• The chemicals should be purchased from reliable source.
• The compounds used should be chemically pure and weighing should be accurate.
• The triple glass distilled water or Millipore water should be used for dissolving the salts.
• Completely dissolve one salt before adding another salt.
• The buffer solution should be sterilized by autoclaving before use.

1. Preparation of Buffer

Materials Required

• Triple distillation water/Millipore water
• Chemicals (Tris, Citric acid monohydrate, Fructose)
• Glycerol
• Weigh the chemicals as per the requirement given above for preparation of 1 litre diluent

Buffer Preparation (for 1 Litre Diluent)

• Take the triple distilled water in a sterile beaker according to the requirement
• Weigh the chemicals accurately
• Add the chemicals one by one and dissolve it by magnetic stirrer by required speed
• Finally add the glycerol at 7% level i.e. 70 ml
• Make the volume up to 800 ml by adding distilled water
• Completely mix the glycerol by magnetic stirrer for at least 20 minutes
• Care should be taken while stirring that air bubbles should not be formed by over speed of the stirrer.
• The pH of buffer should be 6.8-6.9.

Sterilization of Buffer

• The prepared buffer is sterilized by autoclaving at 100:C for 15 min at 5 psi.
• Cool the buffer to room temperature
• Generally the buffer is prepared on previous day evening, sterilized and kept under laminar air flow over night.
• The diluent is prepared by adding egg yolk in the morning immediately before semen collection.

2. Preparation of Diluents

• The egg yolk is prepared as per the standard procedure
• For 1 litre of diluent, 200 ml of egg yolk is required
• The egg yolk is added and mixed by using magnetic stirrer
• The antibiotics are also added while stirring with egg yolk
• Care should be taken while stirring that air bubbles should not be formed by over speed of the stirrer.
• The pH of the diluents should be 6.7-6.8.

Calculation of Dilution Rate

For French Medium Straw

The French medium straw is having 20 million sperms in 0.50 ml.

The total diluted volume is 62.5 ml which includes the semen volume also. So to the 5 ml semen 57.5 ml diluents is added to make the final volume of 62.5 ml.

For French Mini Straw

The French min straw is having 20 million sperms in 0.25 ml.

The total diluted volume is 125 ml which includes the semen volume also. So to the 5 ml semen 120 ml diluents is added to make the final volume of 125 ml.

Dilution of Semen

Immediately after semen collection the volume, concentration and initial motility are estimated.

A minimum of 2.5 ml volume, 500 million concentration and 70% initial motility is required to process a semen sample.

The semen is diluted initially with 1:1 dilution for assessing the initial motility. Then the dilution rate is calculated as per the above method (depends upon the packing volume) and the final dilution is made in a sterile beaker.

While diluting the semen the diluents should not be poured directly over the semen. It should be poured in the side walls of the beaker without air bubble formation.